Helen McNally

Acrolein-Mediated Mechanisms of Neuronal Death

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 μM acrolein caused a dramatic change in cell morphology as early as 4 hr. Cytoskeletal structures significantly deteriorated after exposure to 100 μM acrolein as demonstrated by fluorescence microscopy, whereas calpain activity increased significantly at this concentration. Cell viability assays indicated significant cell death with 100 μM acrolein by 4 hr. Caspase 3 activity and DNA fragmentation assays were performed and supported the notion that 100 μM acrolein induced PC12 cell death by the mechanism of necrosis, not apoptosis.

Self-assembly of micro- and nano-scale particles using bio-inspired events

The head and foot ends of a mattress supporting frame or operating table are articulately connected with the piston rods of two hydraulic motors whose double-acting cylinders are articulately connected to a shiftable bottom frame. The lower chambers of the cylinders can be connected with a supply conduit for pressurized fluid by way of discrete working conduits and discrete solenoid operated directional control valves. The upper chambers of the two cylinders are connected to each other by a first control conduit and the first control conduit is connected with the working conduit for the cylinder at the head end by means of a second control conduit. The supply conduit receives pressurized fluid from a single pump and each valve is further connected with a return conduit for spent fluid. The valve members of the two valves are movable independently of each other between first or neutral positions in which they seal the supply conduit and the return conduit from the respective working conduits, second positions in which they connect the supply conduit with the respective working conduits, and third positions in which they connect the return conduit with the respective working conduits. A solenoid operated shutoff valve can be installed in the working conduit for the cylinder at the head end to seal the lower chamber of such cylinder from the supply and return conduits as well as from the respective working conduit and both control conduits when the foot end is to be raised or lowered without any changes in the level of the head end. The single pump can supply pressurized fluid to the motors for one or more additional frames or tables as well as to auxiliary cylinders which can move the head rest and/or the foot rest of a frame or table between different levels and/or different positions of inclination.

Development and evaluation of transferrin-stabilized paclitaxel nanocrystal formulation

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 3months 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 12days post initial dosing. However, the paclitaxel-transferrin formulation showed a lower level of toxicity, which is indicated by a 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.

Enhanced neurite alignment on micro-patterned poly-L-lactic acid films

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.

Comparative three-dimensional imaging of living neurons with confocal and atomic force microscopy.

Atomic 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.

Neuronal elasticity as measured by atomic force microscopy

A cells form and function is determined to a great extent by its cellular membrane and the underlying cytoskeleton. Understanding changes in the cellular membrane and cytoskeleton can provide insight into aging and disease of the cell. The atomic force microscope (AFM) allows unparalled resolution for the imaging of these cellular components and the ability to probe their mechanical properties. This report describes our progress toward the use of AFM as a tool in neuroscience applications. Elasticity measurements are reported on living chick embryo dorsal root ganglion and sympathetic neurons in vitro. The neuronal cellular body and growth cones regions are examined for variations in cellular maturity. In addition, cellular changes due to exposure to various environmental conditions and neurotoxins are investigated. This report includes data obtained on different AFM systems, using various AFM techniques and thus also provides knowledge of AFM instruments and methodology.

Imaging and manipulating living neurons with atomic force microscopy

Atomic force microscopy (AFM) applications in medicine and biology promises to be significant. Resolutions of living biological materials provided by this technology in the native environment far surpass any modality currently available. The AFM can also be used to physically interact with the sample of interest, allowing for novel experimentation. This report discusses three-dimensional architectures of living chick dorsal root ganglion and sympathetic ganglion somas and growth cones. Secondly, the AFM has been used to inflict damage to these neurons and subsequently image the cells response to injury. In the Center for Paralysis Research, we intend to expand on these preliminary investigations toward a better understanding of neurotrauma and nerve repair.

Electric field and Charged Molecules Mediated Self-Assembly for Electronic Devices

In this paper we present techniques, utilizing dielectrophoresis and electrohydrodynamics, which can possibly be used for assembling devices suspended in a solution onto a binding site on a substrate. We explored the concepts using micro-scale negatively charged polystyrene beads and rectangular silicon blocks. Dielectrophoretic forces on devices in buffer solutions were examined as a function of frequency of the applied AC signal. The observed results can be explained by taking in account electro-thermal and AC electroosmotic effects. The study described in the paper can be used for placing and assembling micro and nano-electronic devices and objects at specific sites on various substrates, in combination with bio-inspired biological binding techniques such as DNA hybridization, antigen-antibody interactions, and ligand-receptor (avidin-biotin) interactions.

Maximizing Nanotechnology Education at Purdue University: Its Integration into the Electrical Engineering Technology Curriculum

Nanotechnology education continues to be a national priority [5], [12] while requiring an integration of the basic disciplines (physics, chemistry, and biology) into the systems of engineering and technology. Training the next-generation workforce in this complex and highly interdisciplinary field presents many opportunities and challenges. Various nanotechnology courses and programs [1], [4], [15] have been developed with differing levels of success. The issues of prerequisite material, student selective interests, and program-specific requirements are a few of the challenges that have slowed the rate of nanotechnology education. Also, access to and training on scanning probe microscopes (SPMs), one of the premier tools of nanotechnology [2], has been limited, particularly at the undergraduate level. A few laboratories [7], [13] have been centered around the atomic force microscope (AFM), a subset of the SPM. However, these systems are expensive, and it is a challenge to retain the expertise necessary to maintain, operate, and train students on these systems.

Basic: B io-Inspired A ssembly of S emiconductor I ntegrated C ircuits

In the recent years, biologically-inspired self-assembly of artificial structures, some with useful optical properties, has been demonstrated. However, to date there has been no demonstration of self-assembly of useful electronic devices for the construction of complex systems. In this paper, a new process called BASIC (Bio-Inspired Assembly of Semiconductor Integrated Circuits) is proposed. The main theme is to use the mutual binding (hybridization) and specificity of DNA strands (oligonucleotides) for the assembly of useful silicon devices on silicon or other substrate. These devices need to be ‘released’ from their host substrate into a liquid medium where they can be functionalized with single stranded DNA. Silicon-on-insulator (SOI) substrates, which naturally lend themselves for such application, due to the presence of an oxide layer underlying the silicon layer, are used. These devices can vary in size and have a thin gold layer on one surface. This approach can be used to assemble micro and nano-scale devices and circuits and can also be a powerful technique for heterogeneous integration of materials (e.g. Si on Glass or polymer). The general idea of the BASIC process can also be extended to be used with any antibody/antigen complex. Preliminary results regarding the fabrication and release of the device islands will be presented. In addition, surface AFM characterization of the gold surfaces, prior to attachment of bio-molecules, is also presented.