It is well known that traumatic injury in the central nervous system can be viewed as a primary injury and a secondary injury. Increases in oxidative stress lead to breakdown of membrane lipids (lipid peroxidation) during secondary injury. Acrolein, an alpha,beta-unsaturated aldehyde, together with other aldehydes, increases as a result of self-propagating lipid peroxidation. Historically, most research on the pathology of secondary injury has focused on reactive oxygen species (ROS) rather than lipid peroxidation products. Little is known about the toxicology and cell death mediated by these aldehydes. In this study, we investigated and characterized certain features of cell death induced by acrolein on PC12 cells as well as cells from dorsal root ganglion (DRG) and sympathetic ganglion in vitro. In the companion paper, we evaluated a possible means to interfere with this toxicity by application of a compound that can bind to and inactivate acrolein. Here we use both light and atomic force microscopy to study cell morphology after exposure to acrolein. Administration of 100 microM acrolein caused a dramatic change in cell morphology as early as 4 hr. Cytoskeletal structures significantly deteriorated after exposure to 100 microM acrolein as demonstrated by fluorescence microscopy, whereas calpain activity increased significantly at this concentration. Cell viability assays indicated significant cell death with 100 microM acrolein by 4 hr. Caspase 3 activity and DNA fragmentation assays were performed and supported the notion that 100 microM acrolein induced PC12 cell death by the mechanism of necrosis, not apoptosis.
The aim of the present study was to prepare and evaluate a paclitaxel nanocrystal-based formulation stabilized by serum protein transferrin in a non-covalent manner. The pure paclitaxel nanocrystals were first prepared using an antisolvent precipitation method augmented by sonication. The serum protein transferrin was selected for use after evaluating the stabilizing effect of several serum proteins including albumin and immunoglobulin G. The formulation contained approximately 55~60% drug and was stable for at least 3 months at 4 °C. In vivo antitumor efficacy studies using mice inoculated with KB cells demonstrate significantly higher tumor inhibition rate of 45.1% for paclitaxel-transferrin formulation compared to 28.8% for paclitaxel nanosuspension treatment alone. Interestingly, the Taxol® formulation showed higher antitumor activity than the paclitaxel-transferrin formulation, achieving a 93.3% tumor inhibition rate 12 days post initial dosing. However, the paclitaxel-transferrin formulation showed a lower level of toxicity, which is indicated by steady increase in body weight of mice over the treatment period. In comparison, treatment with Taxol® resulted in toxicity issues as body weight decreased. These results suggest the potential benefit of using a serum protein in a non-covalent manner in conjunction with paclitaxel nanocrystals as a promising drug delivery model for anticancer therapy.
Atomic Force Microscopy (AFM) has been used to image the morphology of developing neurons and their processes. Additionally, AFM can physically interact with the cell under investigation in numerous ways. Here we use the AFM to both three-dimensionally image the neuron and to inflict a nano/micro-puncture to its membrane. Thus, the same instrument used as a tool to precisely penetrate/cut the membrane at the nanoscale level is employed to image the morphological responses to damage. These first high resolution AFM images of living chick dorsal root ganglion cells and cells of sympathetic ganglion and their growing processes provide confirmation of familiar morphologies. The increased resolution of the AFM revealed these structures to be significantly more complex and variable than anticipated. Moreover we describe novel, dynamic, and unreported architectures, particularly large dorsally projecting ridges, spines, and ribbons of cytoplasm that appear and disappear on the order of minutes. In addition, minute (ca. 100 nm) hair-like extensions of membrane along the walls of nerve processes that also shift in shape and density, appearing and disappearing over periods of minutes were seen. We also provide "real time'' images of the death of the neuron cell body after nano/micro scale damage to its membrane. These somas excreted their degraded cytoplasm, revealed as an enlarging pool beneath and around the cell. Conversely, identical injury, even repeated perforations and nanoslices, to the neurite's membrane do not lead to demise of the process. This experimental study not only provides unreported neurobiology and neurotrauma, but also emphasizes the unique versatility of AFM as an instrument that can (1) physically manipulate cells, (2) provide precise quantitative measurements of distance, surface area and volume at the nanoscale if required, (3) derive physiologically significant data such as membrane pressure and compliance, and (4) during the same period of study-provide unexcelled imaging of living samples.
Comparative three-dimensional imaging of living neurons with confocal and atomic force microscopy" (2005 AbstractAtomic force microscopy applications extend across a number of fields; however, limitations have reduced its effectiveness in live cell analysis. This report discusses the use of AFM to evaluate the three-dimensional (3-D) architecture of living chick dorsal root ganglia and sympathetic ganglia. These data sets were compared to similar images acquired with confocal laser scanning microscopy of identical cells. For this comparison we made use of visualization techniques which were applicable to both sets of data and identified several issues when coupling these technologies. These direct comparisons offer quantitative validation and confirmation of the character of novel images acquired by AFM. This paper is one in a series emphasizing various new applications of AFM in neurobiology.
The ability of the damaged central nervous system and peripheral nervous system to properly recover hinges on the regenerative mechanisms and functional reconnection to appropriate targets. Successful pathfinding of axons is controlled by a complex interplay of diffusible or substrate-bound biochemical and electrical cues. Physical guidance has also been shown to occur in vivo and in vitro, either via cell-cell or cell-extracellular matrix mediated contact. In the current study, we probe the role of contact guidance in facilitating neural regeneration and pathfinding. Using soft lithographic techniques, we have created thin films of poly-L-lactic acid polymer (PLLA) possessing periodic features approaching the nanometer regime. Rat PC-12 cells and chick sympathetic neurons were subsequently cultured onto these substrates and parameters, such as neurite emergence and orientation angle, neurite length, and neuronal architecture are characterized. Our results reveal that both PC-12 and chick sympathetic neurites can be effectively guided by unidirectional grooves as small as 100 nm in height and 1 microm in width. Moreover, sympathetic cells produced neurites that were longer on patterned substrata than on controls. The development of novel degradable micro/nanopatterned substrates for cell study will permit more in-depth analysis of contact mediated guidance mechanisms in addition to having applications in neural and tissue engineering.
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