RESUMEN
The act of defining neuropoietic progenitor/stem cells is still in its early phases. Epidermal growth factor (EGF) stimulates extended proliferation of aggregates of subventricular striatal cells, taken from E15 mouse striatum, termed neurospheres in liquid culture. We have shown here and in previous work, using either immunohistochemistry or RT-PCR, that neurosphere cells express 13 cytokines (32 tested) and 20 cytokine receptors (28 tested), with 11 potential paracrine and nine potential autocrine loops. The neurotrophin receptors, Trk A, B, and C, were all expressed. Using a newly developed FACS single cell deposition technique, we evaluated the capacity of single EGF stimulated neurosphere cells to respond to the ligands for Trk A and B, nerve growth factor (NGF), and brain-derived neurotrophin factor (BDNF). Addition of NGF or BDNF to EGF for 14 days had no effect, but removal of EGF at day 14 with subsequent addition of BDNF or NGF resulted in an increase in neuronal and astroglial, but not oligodendrocyte, colony cells at 21 and 28 days of culture for BDNF, and of both cell types at 28 days for NGF. Tri-lineage colonies increased at day 21 with BDNF and at day 28 for both NGF and BDNF. Gross colony morphology also showed changes with neurotrophin addition, forming multiple individual cell balls or filamentous spreads. When EGF was withdrawn, a threshold effect was observed, with small, but not large, colonies ceasing growth. BDNF and NGF showed no effects on cell proliferation when compared to EGF controls, as determined by 5'-bromo-2-deoxyuridine (BrdU) incorporation and thus, they appear to affect differentiation of progenitor cells. These data indicate a sequential action of cytokines with EGF maintaining viability and proliferation and blocking differentiation. Removal of EGF is then permissive for the differentiating effects of BDNF and NGF. These data further indicate that the majority of EGF neurosphere clones have neurotrophin dependent tri-lineage potential.
Asunto(s)
Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Bromodesoxiuridina/farmacología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Citocinas/genética , Citocinas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Feto , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
A previous investigation suggested that striation patterns spanning individual muscles in longitudinally oriented MR images may represent the orientation of its fascicles. In this study, we confirmed that these striation patterns could be used to infer fascicle orientation and to compute other architectural features of muscles from MR images. The volumes of 14 muscles within a cadaveric thigh were shown to be estimated accurately from cross-sectional MR images by comparison with direct measures from muscle mass. The angles of striations were measured at several positions within vastus medialis and semimembranosus from sagittal and frontal-plane MR images. Mathematical techniques were developed to infer the 3-dimensional orientation of fascicles based on these striation angles. The angle of striations in a 3rd oblique plane was shown to agree with mathematical predictions based on these computed orientations. The pennation angle, defined as the angle between the fascicles and the line of action of the muscle, predicted from the MR images, was similar to directly measured values. Interestingly, the pennation angle of these fascicles varied along the length of the muscle; in vastus medialis, pennation angle ranged from 5 degrees to 50 degrees in a proximodistal direction. Procedures were developed and validated to compute fascicle length by projection of fascicle orientation across the 3D shape of the muscles. The use of MR images to estimate muscle morphometry could improve greatly the predictive capabilities of musculoskeletal modelling by reducing the number of unknown model parameters.
Asunto(s)
Imagen por Resonancia Magnética , Músculos/anatomía & histología , Muslo/anatomía & histología , Anciano , Humanos , MasculinoRESUMEN
The present study examined the relative accuracy and precision of MR and CT procedures for determining the CSA of individual muscles from the human thigh. Serial AN, CT and MR cross-sections were obtained from three cadaveric lower limbs. The MR measurements provided accurate and precise values for the CSAs of most thigh muscles, generally within +/- 7.5% of the AN standard. In contrast, CT tended systematically to overestimate the AN CSA by 10-20%. Retest procedures indicated that highly reliable measurements could be obtained from both MR and CT images. However, subjective interpretations of boundaries between closely apposed muscle bellies, particularly for muscles with more than one head, were necessary for resolving entities in the imaging records and this decreased the relative accuracy of MR and CT measures. Interestingly, MR records demonstrated an incomplete septum between vastus lateralis and vastus intermedius more prominently than AN cross-sections. The novel cross-validation procedures used in this study also highlighted several system-based errors in the MR records that, if undetected and left uncorrected, would have seriously biased the morphometric data obtained with this technique. In general, MR provides high resolution images of the human thigh muscles which may be used to obtain valid measures of the CSA of these structures.