An Amperometric Glucose Sensor Integrated into an Insulin Delivery Cannula: In Vitro and In Vivo Evaluation.

BACKGROUND: Labeling prohibits delivery of insulin at the site of subcutaneous continuous glucose monitoring (CGM). Integration of the sensing and insulin delivery functions into a single device would likely increase the usage of CGM in persons with type 1 diabetes. METHODS: To understand the nature of such interference, we measured glucose at the site of bolus insulin delivery in swine using a flexible electrode strip that was laminated to the outer wall of an insulin delivery cannula. In terms of sensing design, we compared H2O2-measuring sensors biased at 600 mV with redox mediator-type sensors biased at 175 mV. RESULTS: In H2O2-measuring sensors, but not in sensors with redox-mediated chemistry, a spurious rise in current was seen after insulin lis-pro boluses. This prolonged artifact was accompanied by electrode poisoning. In redox-mediated sensors, the patterns of sensor signals acquired during delivery of saline and without any liquid delivery were similar to those acquired during insulin delivery. CONCLUSION: Considering in vitro and in vivo findings together, it became clear that the mechanism of interference is the oxidation, at high bias potentials, of phenolic preservatives present in insulin formulations. This effect can be avoided by the use of redox mediator chemistry using a low bias potential.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development.

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aß) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2) that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aß-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta), we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of our double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, our results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.

Fabrication of a Flexible Amperometric Glucose Sensor Using Additive Processes.

This study details the use of printing and other additive processes to fabricate a novel amperometric glucose sensor. The sensor was fabricated using a Au coated 12.7 µm thick polyimide substrate as a starting material, where micro-contact printing, electrochemical plating, chloridization, electrohydrodynamic jet (e-jet) printing, and spin coating were used to pattern, deposit, chloridize, print, and coat functional materials, respectively. We have found that e-jet printing was effective for the deposition and patterning of glucose oxidase inks with lateral feature sizes between ~5 to 1000 µm in width, and that the glucose oxidase was still active after printing. The thickness of the permselective layer was optimized to obtain a linear response for glucose concentrations up to 32 mM and no response to acetaminophen, a common interfering compound, was observed. The use of such thin polyimide substrates allow wrapping of the sensors around catheters with high radius of curvature ~250 µm, where additive and microfabrication methods may allow significant cost reductions.

Can glucose be monitored accurately at the site of subcutaneous insulin delivery?

Because insulin promotes glucose uptake into adipocytes, it has been assumed that during measurement of glucose at the site of insulin delivery, the local glucose level would be much lower than systemic glucose. However, recent investigations challenge this notion. What explanations could account for a reduced local effect of insulin in the subcutaneous space? One explanation is that, in humans, the effect of insulin on adipocytes appears to be small. Another is that insulin monomers and dimers (from hexamer disassociation) might be absorbed into the circulation before they can increase glucose uptake locally. In addition, negative cooperativity of insulin action (a lower than expected effect of very high insulin concentrations)may play a contributing role. Other factors to be considered include dilution of interstitial fluid by the insulin vehicle and the possibility that some of the local decline in glucose might be due to the systemic effect of insulin. With regard to future research, redundant sensing units might be able to quantify the effects of proximity, leading to a compensatory algorithm. In summary, when measured at the site of insulin delivery, the decline in subcutaneous glucose level appears to be minimal, though the literature base is not large. Findings thus far support (1) the development of integrated devices that monitor glucose and deliver insulin and (2) the use of such devices to investigate the relationship between subcutaneous delivery of insulin and its local effects on glucose. A reduction in the number of percutaneous devices needed to manage diabetes would be welcome.

High rate shear strain of three-dimensional neural cell cultures: a new in vitro traumatic brain injury model.

The fidelity of cell culture simulations of traumatic brain injury (TBI) that yield tolerance and mechanistic information relies on both the cellular models and mechanical insult parameters. We have designed and characterized an electro-mechanical cell shearing device in order to produce a controlled high strain rate injury (up to 0.50 strain, 30 s(-1) strain rate) that deforms three-dimensional (3-D) neural cultures (neurons or astrocytes in an extracellular matrix scaffold). Theoretical analysis revealed that these parameters generate a heterogeneous 3-D strain field throughout the cultures that is dependent on initial cell orientation within the matrix, resulting in various combinations of normal and shear strain. The ability to create a linear shear strain field over a range of input parameters was verified by tracking fluorescent microbeads in an acellular matrix during maximal displacement for a range of strains and strain rates. In addition, cell death was demonstrated in rat cortical astrocytes and neurons in response to high rate, high magnitude shear strain. Furthermore, cell response within the 3-D neuronal cultures depended on orientation, with higher predicted shear strain correlating with an increased loss of neurites, indicating that culture configuration may be an important factor in the mechanical, and hence cellular, response to traumatic insults. Collectively, these results suggest that differential responses exist within a 3-D culture subjected to mechanical insult, perhaps mimicking the in vivo environment, and that this new model can be used to investigate the complex cellular mechanisms associated with TBI.

Mechanical stretch to neurons results in a strain rate and magnitude-dependent increase in plasma membrane permeability.

The mechanism by which mechanical impact to brain tissue is transduced to neuronal impairment remains poorly understood. Using an in vitro model of neuronal stretch, we found that mechanical stretch of neurons resulted in a transient plasma membrane permeability increase. Primary cortical neurons, seeded on silicone substrates, were subjected to a defined rate and magnitude strain pulse by stretching the substrates over a fixed cylindrical form. To identify plasma membrane defects, various sized fluorescent molecules were added to the bathing media either immediately before injury or 1, 2, 5, or 10 min after injury and removed one minute later. The percent of cells that took up dye depended on the applied strain rate, strain magnitude and molecular size. Severe stretch (10 sec(-1), 0.30) resulted in significant uptake of all tested molecules (ranging between 0.5 and 8.9 nm radii) with up to 60% of cells positively stained. Furthermore, the neurons remained permeable to the smallest molecule (carboxyfluorescein, 380 Da) up to 5 min after severe stretch but were only permeable to larger molecules (>/=10 kDa) immediately after stretch. These transiently formed membrane defects may be the initiating mechanism that translates mechanical stretch to cellular dysfunction.

Susceptibility of hippocampal neurons to mechanically induced injury.

Experimental models of traumatic cortical brain injury in rodents reveal that specific regions of the hippocampus (e.g., CA3 and hilar subfields) are severely injured despite their distance from the initial insult. Hippocampal neurons may be intrinsically more vulnerable to mechanical insult than cortical neurons due to increased NMDA receptor densities and lower energy capacities, as evidenced by increased susceptibility to ischemic insults. The selective vulnerability of hippocampal neurons was evaluated using an in vitro model of TBI in which either primary rat cortical or hippocampal neurons (E17) seeded onto silicone substrates were subjected to graded levels of mechanical stretch. Although cortical neurons exhibited significantly longer increases in stretch-induced membrane permeability, injury of hippocampal neurons resulted in larger increases in intracellular free calcium concentration [Ca(2+)](i) and cell death. [ATP](i) deficits due to stretch were apparent by 60 min after injury in cortical neurons but recovered by 24 h, whereas significant deficits in [ATP](i) were not observed in hippocampal neurons until 24 h after injury. MK801 pretreatment decreased the stretch-induced [Ca(2+)](i) transients in both hippocampal and cortical cultures, thereby negating the regional specificity. However, MK801 pretreatment did not improve hippocampal viability and paradoxically, significantly increased cell death among cortical neurons. As the hippocampus is the primary brain region responsible for the memory deficits and epileptic seizures associated with TBI, understanding why this region is selectively damaged could lead to the development of more accurate mechanical tolerances as well as effective pharmaceutical agents.