A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and loss of large motor neurons in the spinal cord and brain stem. While much research has focused on mechanisms of motor neuron cell death in the spinal cord, degenerative processes in skeletal muscle and neuromuscular junctions (NMJs) are also observed early in disease development. Although recent studies support the potential therapeutic benefits of targeting the skeletal muscle in ALS, relatively little is known about inflammation and glial responses in skeletal muscle and near NMJs, or how these responses contribute to motor neuron survival, neuromuscular innervation, or motor dysfunction in ALS. We recently showed that human mesenchymal stem cells modified to release glial cell line-derived neurotrophic factor (hMSC-GDNF) extend survival and protect NMJs and motor neurons in SOD1G93A rats when delivered to limb muscles. In this study, we evaluate inflammatory and glial responses near NMJs in the limb muscle collected from a rat model of familial ALS (SOD1G93A transgenic rats) during disease progression and following hMSC-GDNF transplantation. Muscle samples were collected from pre-symptomatic, symptomatic, and end-stage animals. A significant increase in the expression of microglial inflammatory markers (CD11b and CD68) occurred in the skeletal muscle of symptomatic and end-stage SOD1G93A rats. Inflammation was confirmed by ELISA for inflammatory cytokines interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) in muscle homogenates of SOD1G93A rats. Next, we observed active glial responses in the muscle of SOD1G93A rats, specifically near intramuscular axons and NMJs. Interestingly, strong expression of activated glial markers, glial fibrillary acidic protein (GFAP) and nestin, was observed in the areas adjacent to NMJs. Finally, we determined whether ex vivo trophic factor delivery influences inflammation and terminal Schwann cell (TSC) response during ALS. We found that intramuscular transplantation of hMSC-GDNF tended to exhibit less inflammation and significantly maintained TSC association with NMJs. Understanding cellular responses near NMJs is important to identify suitable cellular and molecular targets for novel treatment of ALS and other neuromuscular diseases.

Scattered radiation is a major source of artifacts in flat detector computed tomography (FDCT) due to the increased irradiated volumes. We propose a fast projection-based algorithm for correction of scatter artifacts. The presented algorithm combines a convolution method to determine the spatial distribution of the scatter intensity distribution with an object-size-dependent scaling of the scatter intensity distributions using a priori information generated by Monte Carlo simulations. A projection-based (PBSE) and an image-based (IBSE) strategy for size estimation of the scanned object are presented. Both strategies provide good correction and comparable results; the faster PBSE strategy is recommended. Even with such a fast and simple algorithm that in the PBSE variant does not rely on reconstructed volumes or scatter measurements, it is possible to provide a reasonable scatter correction even for truncated scans. For both simulations and measurements, scatter artifacts were significantly reduced and the algorithm showed stable behavior in the z-direction. For simulated voxelized head, hip and thorax phantoms, a figure of merit Q of 0.82, 0.76 and 0.77 was reached, respectively (Q = 0 for uncorrected, Q = 1 for ideal). For a water phantom with 15 cm diameter, for example, a cupping reduction from 10.8% down to 2.1% was achieved. The performance of the correction method has limitations in the case of measurements using non-ideal detectors, intensity calibration, etc. An iterative approach to overcome most of these limitations was proposed. This approach is based on root finding of a cupping metric and may be useful for other scatter correction methods as well. By this optimization, cupping of the measured water phantom was further reduced down to 0.9%. The algorithm was evaluated on a commercial system including truncated and non-homogeneous clinically relevant objects.

Since the 1980s, theorists have posited that health education that reflects the cultural realities of communities that health educators targeted for behavioral interventions would be more successful than interventions that are not culturally sensitive. Between 1997 and 2002, 52 focus groups of youth, women, and men were conducted in the Appalachian portions of 10 states to discern cultural themes relevant to health education in Appalachia. Groups occurred within the context of 5 studies funded by institutes within the National Institutes of Health. Findings suggest that an emphasis on family shows immense promise as a culturally sensitive approach to health education. Interventions that use the central role of women in the health of their families may be useful. The study results also suggest that one-on-one approaches to health education may prove a promising technique, attacks on individuals and institutions are not useful strategies, and a preference for realism or "the facts" may be a good way to present information.