Label free cell tracking in 3-D tissue engineering constructs with high resolution imaging

Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results

Environmental enrichment facilitates long-term potentiation in embryonic striatal grafts

Background. Housing animals in an enriched environment improves motor and cognitive performance and anatomical connectivity in rodent lesion models of Huntington disease and transplantation of embryonic striatal grafts. Objective. The authors evaluate the extent to which environmental enrichment can modify synaptic plasticity in the host-graft neuronal circuitry to try to find a physiological substrate for the observed improvements. Methods. C57BL/6 mice, housed in enriched or standard environments, received unilateral quinolinic acid lesions of the striatum, followed by embryonic striatal grafts. Then, 3 months posttransplantation, synaptic physiology and plasticity were evaluated by extracellular recording from in vitro striatal slices. Results. Environmental enrichment had no effect on the chance of long-term depression (LTD) induction or expression of LTD from either normal or grafted striatum. In contrast, enrichment increased the chance of long-term potentiation (LTP) induction and level of expression associated with increased levels of brain-derived neurotrophic factor within both the intact and grafted striatum compared with levels in the striatum of animals housed in standard environments. Conclusions. Environmental enrichment induces changes in host-graft corticostriatal LTP, thus providing a potential physiological substrate for the enrichment-induced improvement in motor and cognitive performance. The effect may be mediated by modulation of the trophic environment in which the grafted cells develop and integrate

Identification and characterization of a functional mitochondrial angiotensin system

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging