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The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.
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Catepsina K/antagonistas & inhibidores , Osteoporosis/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Animales , Huesos/diagnóstico por imagen , Radioisótopos de Carbono/química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Femenino , Ligandos , Macaca mulatta , Unión Proteica , Conejos , Radiofármacos/efectos adversos , Radiofármacos/química , Distribución TisularRESUMEN
fMRI can objectively measure pain-related neural activities in humans and animals, providing a valuable tool for studying the mechanisms of nociception and for developing new analgesics. However, due to its extreme sensitivity to subject motion, pain fMRI studies are performed in animals that are immobilized, typically with anesthesia. Since anesthesia could confound the nociceptive processes, it is unknown how well nociceptive-related neural activities measured by fMRI in anesthetized animals correlate with nociceptive behaviors in conscious animals. The threshold to vocalization (VT) in response to an increasing noxious electrical stimulus (NES) was implemented in conscious rats as a behavioral measure of nociception. The antinociceptive effect of systemic (intravenous infusion) lidocaine on NES-induced fMRI signals in anesthetized rats was compared with the corresponding VT in conscious rats. Lidocaine infusion increased VT and suppressed the NES-induced fMRI signals in most activated brain regions. The temporal characteristics of the nociception signal by fMRI and by VT in response to lidocaine infusion were highly correlated with each other, and with the pharmacokinetics (PK) of lidocaine. These results indicate that the fMRI activations in these regions may be used as biomarkers of acute nociception in anesthetized rats. Interestingly, systemic lidocaine had no effect on NES-induced fMRI activations in the primary somatosensory cortex (S1), a result that warrants further investigation.
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Encéfalo/fisiología , Dimensión del Dolor/métodos , Dolor , Anestesia General , Anestésicos Locales/farmacología , Animales , Encéfalo/efectos de los fármacos , Mapeo Encefálico , Estado de Conciencia , Procesamiento de Imagen Asistido por Computador , Lidocaína/farmacología , Imagen por Resonancia Magnética , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Vocalización AnimalRESUMEN
Calcitonin gene-related peptide (CGRP) is a potent neuropeptide whose agonist interaction with the CGRP receptor (CGRP-R) in the periphery promotes vasodilation, neurogenic inflammation and trigeminovascular sensory activation. This process is implicated in the cause of migraine headaches, and CGRP-R antagonists in clinical development have proven effective in treating migraine-related pain in humans. CGRP-R is expressed on blood vessel smooth muscle and sensory trigeminal neurons and fibers in the periphery as well as in the central nervous system. However, it is not clear what role the inhibition of central CGRP-R plays in migraine pain relief. To this end, the CGRP-R positron emission tomography (PET) tracer [(11)C]MK-4232 (2-[(8R)-8-(3,5-difluorophenyl)-6,8-[6-(11)C]dimethyl-10-oxo-6,9-diazaspiro[4.5]decan-9-yl]-N-[(2R)-2'-oxospiro[1,3-dihydroindene-2,3'-1H-pyrrolo[2,3-b]pyridine]-5-yl]acetamide) was discovered and developed for use in clinical PET studies. In rhesus monkeys and humans, [(11)C]MK-4232 displayed rapid brain uptake and a regional brain distribution consistent with the known distribution of CGRP-R. Monkey PET studies with [(11)C]MK-4232 after intravenous dosing with CGRP-R antagonists validated the ability of [(11)C]MK-4232 to detect changes in CGRP-R occupancy in proportion to drug plasma concentration. Application of [(11)C]MK-4232 in human PET studies revealed that telcagepant achieved only low receptor occupancy at an efficacious dose (140 mg PO). Therefore, it is unlikely that antagonism of central CGRP-R is required for migraine efficacy. However, it is not known whether high central CGRP-R antagonism may provide additional therapeutic benefit.
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Acetanilidas/farmacocinética , Analgésicos/farmacocinética , Azepinas/farmacocinética , Encéfalo/metabolismo , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Imidazoles/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Compuestos de Espiro/farmacocinética , Acetanilidas/química , Adulto , Analgésicos/uso terapéutico , Animales , Azepinas/uso terapéutico , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Femenino , Humanos , Imidazoles/uso terapéutico , Macaca mulatta , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/metabolismo , Estructura Molecular , Unión Proteica , Radiofármacos/química , Especificidad de la Especie , Compuestos de Espiro/química , Distribución Tisular , Adulto JovenRESUMEN
This study aims to identify fMRI signatures of nociceptive processing in whole brain of anesthetized rats during noxious electrical stimulation (NES) and noxious mechanical stimulation (NMS) of paw. Activation patterns for NES were mapped with blood oxygen level dependent (BOLD) and cerebral blood volume (CBV) fMRI, respectively, to investigate the spatially-dependent hemodynamic responses during nociception processing. A systematic evaluation of fMRI responses to varying frequencies of electrical stimulus was carried out to optimize the NES protocol. Both BOLD and CBV fMRI showed widespread activations, but with different spatial characteristics. While BOLD and CBV showed well-localized activations in ipsilateral dorsal column nucleus, contralateral primary somatosensory cortex (S1), and bilateral caudate putamen (CPu), CBV fMRI showed additional bilateral activations in the regions of pons, midbrain and thalamus compared to BOLD fMRI. CBV fMRI that offers higher sensitivity compared to BOLD was then used to compare the nociception processing during NES and NMS in the same animal. The activations in most regions were similar. In the medulla, however, NES induced a robust activation in the ipsilateral dorsal column nucleus while NMS showed no activation. This study demonstrates that (1) the hemodynamic response to nociception is spatial-dependent; (2) the widespread activations during nociception in CBV fMRI are similar to what have been observed in (14)C-2-deoxyglucose (2DG) autoradiography and PET; (3) the bilateral activations in the brain originate from the divergence of neural responses at supraspinal level; and (4) the similarity of activation patterns suggests that nociceptive processing in rats is similar during NES and NMS.
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Encéfalo/fisiopatología , Circulación Cerebrovascular , Estimulación Eléctrica , Imagen por Resonancia Magnética/métodos , Red Nerviosa/fisiopatología , Dolor/fisiopatología , Estimulación Física , Animales , Velocidad del Flujo Sanguíneo , Volumen Sanguíneo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Glycine transporter 1 (GlyT1) inhibitors have emerged as potential treatments for schizophrenia due to their potentiation of NMDA receptor activity by modulating the local concentrations of the NMDA co-agonist glycine. [18F]MK-6577 is a potent and selective GlyT1 inhibitor PET tracer. Although differences in ligand kinetics can be expected between non-human primates and humans, the tracer pre-clinical evaluation can provide valuable information supporting protocol design and quantification in the clinical space. The main objective of this work was to evaluate the in vivo kinetics of [18F]MK-6577 in rhesus monkey brain. Additionally, a method for estimating the tracer input function from the tracer brain tissue kinetics and venous sampling was validated. This technique was applied for determination of the dose-occupancy relationship of a GlyT1 inhibitor in monkey brain. METHODS: Compartmental and Logan graphical analysis were utilized for quantification of the [18F]MK-6577 binding using the measured tracer arterial input function. The stability of the tracer volume of distribution relative to scan length was assessed. The proposed model-based input function method takes advantage of the agreement between the tracer concentration in arterial and venous plasma from ~5 min. The approach estimates the initial peak of the input curve by adding a gamma like function term to the measured venous curve. The parameters of the model function were estimated by simultaneously fitting several brain time activity curves to a compartmental model. RESULTS: Good agreement was found between the model-based and the measured arterial plasma curve and the corresponding distribution volumes. The Logan analysis was the preferred method of analysis providing reliable and stable volume of distribution and occupancy results using a 90 and possibly 60 min scan length. CONCLUSION: The model-based input function method and Logan analysis are well suited for quantification of [18F]MK-6577 binding and GlyT1 occupancy in monkey brain.
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Química Encefálica/fisiología , Encéfalo/diagnóstico por imagen , Agonistas de Aminoácidos Excitadores , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Algoritmos , Animales , Cuerpo Estriado/diagnóstico por imagen , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacocinética , Radioisótopos de Flúor , Proteínas de Transporte de Glicina en la Membrana Plasmática/análisis , Procesamiento de Imagen Asistido por Computador , Macaca mulatta , Modelos Neurológicos , Modelos Estadísticos , Tomografía de Emisión de Positrones , Receptores de N-Metil-D-Aspartato/fisiología , Tálamo/diagnóstico por imagenRESUMEN
Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.
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Encéfalo/diagnóstico por imagen , Radioisótopos de Flúor/farmacocinética , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Receptores de Neuropéptido Y/biosíntesis , Animales , Autorradiografía , Humanos , Macaca mulatta , Tomografía de Emisión de Positrones , Trazadores RadiactivosRESUMEN
Two moderately lipophilic, high affinity ligands for metabotropic glutamate receptor subtype 1 (mGluR1) were radiolabeled with a positron-emitting radioisotope and evaluated in rhesus monkey as potential PET tracers. Both ligands were radiolabeled with fluorine-18 via nucleophilic displacement of the corresponding 2-chloropyridine precursor with [¹8F]potassium fluoride. [¹8F]MK-1312 was found to have a suitable signal for quantification of mGluR1 receptors in nonhuman primates and was more thoroughly characterized. In vitro autoradiographic studies with [¹8F]MK-1312 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the known distribution of mGluR1, with the highest uptake in the cerebellum, moderate uptake in the hippocampus, thalamus, and cortical regions, and lowest uptake in the caudate and putamen. In vitro saturation binding studies in rhesus monkey and human cerebellum homogenates confirmed that [¹8F]MK-1312 binds to a single site with a B(max) /K(d) ratio of 132 and 98, respectively. PET studies in rhesus monkey with [¹8F]MK-1312 showed high brain uptake and a regional distribution consistent with in vitro autoradiography results. Blockade of [¹8F]MK-1312 uptake with mGluR1 allosteric antagonist MK-5435 dose-dependently reduced tracer uptake in all regions of gray matter to a similarly low level of tracer uptake. This revealed a large specific signal useful for determination of mGluR1 receptor occupancy in rhesus monkey. Taken together, these results are promising for clinical PET studies with [¹8F]MK-1312 to determine mGluR1 occupancy of MK-5435.
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Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Fármacos actuantes sobre Aminoácidos Excitadores , Tomografía de Emisión de Positrones , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Autorradiografía/métodos , Sitios de Unión/efectos de los fármacos , Encéfalo/metabolismo , Mapeo Encefálico , Relación Dosis-Respuesta a Droga , Fármacos actuantes sobre Aminoácidos Excitadores/síntesis química , Fármacos actuantes sobre Aminoácidos Excitadores/química , Fármacos actuantes sobre Aminoácidos Excitadores/farmacocinética , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacocinética , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Concentración 50 Inhibidora , Ligandos , Macaca mulatta , Distribución Tisular , Triazoles/síntesis química , Triazoles/farmacocinéticaRESUMEN
Objective measure of pain is valuable in drug discovery research and development of analgesics. Spinal cord is an important relay of the pain pathway, and fMRI offers an excellent opportunity to quantify pain using activation in the spinal cord induced by painful stimuli. fMRI literature of cervical spinal cord with regard to the spatial extent, in both longitudinal and cross-sectional directions, of neuronal activation induced by noxious stimulation is ambiguous. This study investigates the feasibility of developing a robust pain assay using fMRI in the cervical spinal cord in alpha-chloralose anesthetized rats subjected to transcutaneous noxious electrical stimulation of the forepaw. Blood oxygenation level dependent (BOLD) and blood volume (BV)-weighted fMRI data were acquired without and with intravenous injection of ultra small superparamagnetic iron oxide particles (USPIO), respectively. BOLD data were acquired by gradient-echo (GE) and spin-echo (SE) echo planar imaging (EPI), while BV-weighted fMRI data were acquired only by GE EPI. Cervical spinal cord activity was robustly detected by all three fMRI techniques. The sensitivity of the fMRI signal was highest in GE BV-weighted fMRI followed in order by GE BOLD, and SE BOLD, respectively. Spatially, the fMRI signal extended approximately 9 mm in the longitudinal direction, covering C(4)-C(8) segments, coinciding with the synapse location of afferent terminals from the stimulated site. In the cross-sectional direction, the signal change is localized predominantly to the ipsilateral dorsal region. This study demonstrates that cervical spinal cord fMRI can be performed reliably in anesthetized rats offering it as a potential tool for analgesic drug development.
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Imagen Eco-Planar/métodos , Potenciales Evocados Somatosensoriales , Hierro , Imagen por Resonancia Magnética/métodos , Óxidos , Dolor/fisiopatología , Médula Espinal/patología , Médula Espinal/fisiopatología , Animales , Volumen Sanguíneo , Vértebras Cervicales/patología , Vértebras Cervicales/fisiopatología , Dextranos , Óxido Ferrosoférrico , Nanopartículas de Magnetita , Masculino , Ratas , Ratas WistarRESUMEN
PURPOSE: A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described. PROCEDURES: In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor. RESULTS: [(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships. CONCLUSIONS: [(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.
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Compuestos Heterocíclicos con 2 Anillos/química , Hidrolasas Diéster Fosfóricas/metabolismo , Tomografía de Emisión de Positrones/métodos , Animales , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Femenino , Compuestos Heterocíclicos con 2 Anillos/sangre , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/farmacocinética , Humanos , Macaca mulatta , Masculino , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Factores de TiempoRESUMEN
Conjugation of dextran-coated superparamagnetic iron oxide (SPIO) particles with transactivator protein (Tat)-peptide and fluorescein isothiocyanate (FITC) allows cells to readily uptake SPIO particles. This makes possible high-resolution, real-time imaging of these cells by magnetic resonance imaging (MRI). First, we need to understand how various subpopulations take up and maintain SPIO particles. In this report, we have focused on differences in T cells, B cells, and macrophages with respect to cross-linked (CL)-SPIO Tat-FITC particle uptake over 72 hours. We have found that cells quickly take up the particles and that the bead loss that does occur is not related to cell death or apoptosis. In contrast with reports in the literature, we have observed migration of the Tat-peptide conjugates primarily to the cytoplasm rather than the nucleus.
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Fluoresceína-5-Isotiocianato , Productos del Gen rev , Hierro , Imagen por Resonancia Magnética , Óxidos , Bazo/citología , Bazo/fisiología , Animales , Movimiento Celular , Reactivos de Enlaces Cruzados , Dextranos , Estabilidad de Medicamentos , Femenino , Óxido Ferrosoférrico , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacocinética , Productos del Gen rev/química , Productos del Gen rev/farmacocinética , Hierro/química , Hierro/farmacocinética , Nanopartículas de Magnetita , Masculino , Ratones , Ratones Endogámicos , Óxidos/química , Óxidos/farmacocinética , Ratas , Ratas Endogámicas , Bazo/metabolismoRESUMEN
This paper addresses the problem of enhancing spatiotemporal resolution of ultra-small superparamagnetic iron oxide (USPIO)-enhanced dynamic MRI of rat kidneys. To alleviate the limited resolution problem of conventional full-scan Fourier imaging methods, we use a generalized series-based imaging scheme to reduce coverage of kappa-space. Experimental results demonstrate that the generalized series imaging method with basis functions constructed using two references (pre- and post-contrast) can reduce the number of phase encodings measured during the dynamic contrast wash-in process by a factor of 4 with a negligible or minimal loss of image quality. The method is expected to make 3D studies possible using USPIO-enhanced dynamic imaging of rat kidneys, and prove valuable for early detection of renal rejection after kidney transplantation.
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Hierro , Riñón/anatomía & histología , Imagen por Resonancia Magnética/métodos , Óxidos , Animales , Medios de Contraste , Dextranos , Óxido Ferrosoférrico , Rechazo de Injerto/diagnóstico , Procesamiento de Imagen Asistido por Computador , Trasplante de Riñón/patología , Nanopartículas de Magnetita , RatasRESUMEN
Rational modification of the potent calcitonin gene-related peptide (CGRP) receptor antagonist MK-3207 led to a series of analogues with enhanced CNS penetrance and a convenient chemical handle for introduction of a radiolabel. A number of (11)C-tracers were synthesized and evaluated in vivo, leading to the identification of [(11)C]8 ([(11)C]MK-4232), the first positron emission tomography tracer for the CGRP receptor.
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We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.
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Spinal cord fMRI offers an excellent opportunity to quantify nociception using neuronal activation induced by painful stimuli. Measurement of the magnitude of stimulation-induced activation, and its suppression with analgesics can provide objective measures of pain and efficacy of analgesics. This study investigates the feasibility of using spinal cord fMRI in anesthetized rats as a pain assay to test the analgesic effect of locally and systemically administered lidocaine. Blood volume (BV)-weighted fMRI signal acquired after intravenous injection of ultrasmall superparamagnetic iron oxide (USPIO) particles was used as an indirect readout of the neuronal activity. Transcutaneous noxious electrical stimulation was used as the pain model. BV-weighted fMRI signal could be robustly quantified on a run-by-run basis, opening the possibility of measuring pharmacodynamics (PD) of the analgesics with a temporal resolution of approximately 2 min. Local administration of lidocaine was shown to ablate all stimulation-induced fMRI signals by the total blockage of peripheral nerve transmission, while the analgesic effect of systemically administered lidocaine was robustly detected after intravenous infusion of approximately 3mg/kg, which is similar to clinical dosage for human. This study establishes spinal cord fMRI as a viable assay for analgesics. With respect to the mode of action of lidocaine, this study suggests that systemic lidocaine, which is clinically used for the treatment of neuropathic pain, and believed to only block the peripheral nerve transmission of abnormal neural activity (ectopic discharge) originating from the damaged peripheral nerves, also blocks the peripheral nerve transmission of normal neural activity induced by transcutaneous noxious electrical stimulation.
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Anestésicos Locales/administración & dosificación , Lidocaína/administración & dosificación , Dolor/patología , Médula Espinal/irrigación sanguínea , Médula Espinal/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Estimulación Eléctrica/efectos adversos , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Oxígeno/sangre , Dolor/tratamiento farmacológico , Dolor/etiología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Factores de TiempoRESUMEN
Spinal cord fMRI is a useful tool for studying spinal mechanisms of pain, hence for analgesic drug development. Its technical feasibility in both humans and rats has been demonstrated. This study investigates the reproducibility, robustness, and spatial accuracy of fMRI of lumbar spinal cord activation due to transcutaneous noxious and non-noxious electrical stimulation of the hindpaw in alpha-chloralose-anesthetized rats. Blood oxygenation level-dependent (BOLD) and blood volume-weighted fMRI data were acquired without and with intravenous injection of ultra small superparamagnetic iron oxide particles (USPIO), respectively, using a gradient echo (GE) echo planar imaging (EPI) technique at 4.7 T. Neuronal activation in the spinal cord induced by noxious stimulation to the hindpaw (2 ms wide, 5 mA amplitude, known to activate C-fibers) can be robustly detected by both fMRI techniques with excellent reproducibility and peaked at the stimulus frequency of 40 Hz. However, both fMRI techniques were not sensitive to neuronal activation in spinal cord induced by non-noxious stimulation (0.3 ms, 1.5 mA, known only to activate A-fibers). Spatially, the fMRI signal extended approximately 5 mm in the longitudinal direction, covering L(3)-L(5) segments. In the cross-sectional direction, the highest signal change of blood volume-weighted fMRI was in the middle of the ipsilateral dorsal horn, which roughly corresponds to laminae V and VI, while the highest signal change of BOLD fMRI was in the ipsilateral dorsal surface. This study demonstrates that spinal cord fMRI can be performed in anesthetized rats reliably and reproducibly offering it as a potential tool for analgesic drug discovery.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Oxígeno/sangre , Dolor/patología , Médula Espinal/patología , Animales , Medios de Contraste , Interpretación Estadística de Datos , Imagen Eco-Planar , Estimulación Eléctrica , Compuestos Férricos , Pie , Miembro Posterior , Magnetismo , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Magnetic resonance imaging (MRI) for non-invasively detecting renal rejection was developed by monitoring the accumulation of macrophages labeled with dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles at the rat renal allografts during acute rejection. METHODS: Five groups of male rats with DA-->BN renal allografts and one group with BN-->BN renal isografts were investigated by MRI before, immediately after, and 24 hr after intravenous infusion with different doses of USPIO particles. All infusions were done on post-operative day 4. MRI experiments were carried out in a 4.7-Tesla instrument using a gradient echo sequence. RESULTS: MR signal intensity (MRSI) of the cortex was found to decrease with higher dosages of USPIO particles. In the absence of USPIO infusion, a decrease in MRSI was seen in the medulla region, presumably due to hemorrhage associated with renal graft rejection, while no significant change was observed in the cortex. The optimal dose of USPIO particles for visualizing rejection-associated changes in our rat kidney model appears to be 6 mg Fe/kg body weight. Iron staining results correlated with the MRSI data, indicating that the signal reduction in the MR images was due to the presence of iron. Immunohistochemical results indicated that USPIO particles were mostly taken up by infiltrating macrophages in the rejecting grafts. CONCLUSIONS: Our results suggest that MRI with intravenous administration of dextran-coated USPIO particles appears to be a valuable and promising tool that can be used as a non-invasive and sensitive method to detect graft rejection in renal transplantation.