Maribel Vazquez

Bio-heat transfer model of deep brain stimulation-induced temperature changes

There is a growing interest in the use of chronic deep brain stimulation (DBS) for the treatment of medically refractory movement disorders and other neurological and psychiatric conditions. Fundamental questions remain about the physiologic effects and safety of DBS. Previous basic research studies have focused on the direct polarization of neuronal membranes by electrical stimulation. The goal of this paper is to provide information on the thermal effects of DBS using finite element models to investigate the magnitude and spatial distribution of DBS induced temperature changes. The parameters investigated include: stimulation waveform, lead selection, brain tissue electrical and thermal conductivity, blood perfusion, metabolic heat generation during the stimulation. Our results show that clinical deep brain stimulation protocols will increase the temperature of surrounding tissue by up to 0.8 deg C depending on stimulation/tissue parameters.

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.

A Microfluidic Device to Establish Concentration Gradients Using Reagent Density Differences

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.

Microfluidic Generated EGF-Gradients Induce Chemokinesis of Transplantable Retinal Progenitor Cells via the JAK/STAT and PI3Kinase Signaling Pathways

A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.

Bio-heat transfer model of deep brain stimulation induced temperature changes.

There is a growing interest in the use of chronic deep brain stimulation (DBS) for the treatment of medically refractory movement disorders and other neurological and psychiatric conditions. Fundamental questions remain about the physiologic effects and safety of DBS. Previous basic research studies have focused on the direct polarization of neuronal membranes by electrical stimulation. The goal of this paper is to provide information on the thermal effects of DBS using finite element models to investigate the magnitude and spatial distribution of DBS induced temperature changes. The parameters investigated include: stimulation waveform, lead selection, brain tissue electrical and thermal conductivity, blood perfusion, metabolic heat generation during the stimulation. Our results show that clinical deep brain stimulation protocols will increase the temperature of surrounding tissue by up to 0.8degC depending on stimulation/tissue parameters

Targeted extracellular nanoparticles enable intracellular detection of activated epidermal growth factor receptor in living brain cancer cells

UNLABELLED Mechanistic study of biological processes via Quantum Dots (QDs) remain constrained by inefficient QD delivery methods and consequent altered cell function. Here the authors present a rapid method to label activated receptor populations in live cancer cells derived from medulloblastoma and glioma tumors. The authors used QDs to bind the extracellular domain of Epidermal Growth Factor Receptor (EGF-R) proteins and then induced receptor activation to facilitate specific detection of intracellular, activated EGF-R subpopulations. Such labeling enables rapid identification of biological markers characteristic of tumor type, grade and chemotherapy resistance. FROM THE CLINICAL EDITOR In this paper, a rapid, quantum dot-based method is presented with the goal of labeling activated receptor populations in live cancer cells. More accurate characterization of medulloblastoma and glioma cancer cells using this biomarker detection technique may lead to a more specific targeted therapy.

Sendai virus-based liposomes enable targeted cytosolic delivery of nanoparticles in brain tumor-derived cells

ABSTRACTBackgroundNanotechnology-based bioassays that detect the presence and/or absence of a combination of cell markers are increasingly used to identify stem or progenitor cells, assess cell heterogeneity, and evaluate tumor malignancy and/or chemoresistance. Delivery methods that enable nanoparticles to rapidly detect emerging, intracellular markers within cell clusters of biopsies will greatly aid in tumor characterization, analysis of functional state and development of treatment regimens.ResultsExperiments utilized the Sendai virus to achieve in vitro, cytosolic delivery of Quantum dots in cells cultured from Human brain tumors. Using fluorescence microscopy and Transmission Electron Microscopy, in vitro experiments illustrated that these virus-based liposomes decreased the amount of non-specifically endocytosed nanoparticles by 50% in the Human glioblastoma and medulloblastoma samples studied. Significantly, virus-based liposome delivery also facilitated targeted binding of Quantum dots to cytosolic Epidermal Growth Factor Receptor within cultured cells, focal to the early detection and characterization of malignant brain tumors.ConclusionsThese findings are the first to utilize the Sendai virus to achieve cytosolic, targeted intracellular binding of Qdots within Human brain tumor cells. The results are significant to the continued applicability of nanoparticles used for the molecular labeling of cancer cells to determine tumor heterogeneity, grade, and chemotherapeutic resistivity.

Low Concentration Microenvironments Enhance the Migration of Neonatal Cells of Glial Lineage

Glial tumors have demonstrated abilities to sustain growth via recruitment of glial progenitor cells (GPCs), which is believed to be driven by chemotactic cues. Previous studies have illustrated that mouse GPCs of different genetic backgrounds are able to replicate the dispersion pattern seen in the human disease. How GPCs with genetic backgrounds transformed by tumor paracrine signaling respond to extracellular cues via migration is largely unexplored, and remains a limiting factor in utilizing GPCs as therapeutic targets. In this study, we utilized a microfluidic device to examine the chemotaxis of three genetically-altered mouse GPC populations towards tumor conditioned media, as well as towards three growth factors known to initiate the chemotaxis of cells excised from glial tumors: Hepatocyte Growth Factor (HGF), Platelet-Derived Growth Factor-BB (PDGF-BB), and Transforming Growth Factor-α (TGF-α). Our results illustrate that GPC types studied exhibited chemoattraction and chemorepulsion by different concentrations of the same ligand, as well as enhanced migration in the presence of ultra-low ligand concentrations within environments of high concentration gradient. These findings contribute towards our understanding of the causative and supportive roles that GPCs play in tumor growth and reoccurrence, and also point to GPCs as potential therapeutic targets for glioma treatment.

A model microfluidics-based system for the human and mouse retina.

The application of microfluidics technologies to the study of retinal function and response holds great promise for development of new and improved treatments for patients with degenerative retinal diseases. Restoration of vision via retinal transplantation therapy has been severely limited by the low numbers of motile cells observed post transplantation. Using modern soft lithographic techniques, we have developed the μRetina, a novel and convenient biomimetic microfluidics device capable of examing the migratory behavior of retinal lineage cells within biomimetic geometries of the human and mouse retina. Coupled computer simulations and experimental validations were used to characterize and confirm the formation of chemical concentration gradients within the μRetina, while real-time images within the device captured radial and theta cell migration in response to concentration gradients of stromal derived factor (SDF-1), a known chemoattractant. Our data underscore how the μRetina can be used to examine the concentration-dependent migration of retinal progenitors in order to enhance current therapies, as well as develop novel migration-targeted treatments.

Electrophoresis using ultra-high voltages.

Optimization of electrophoretic techniques is becoming an increasingly important area of research as microdevices are now routinely adapted for numerous biology and engineering applications. The present work seeks to optimize electrophoresis within microdevices by utilizing ultra-high voltages to increase sample concentration prior to separation. By imaging fluorescently-tagged DNA samples, the effects of both conventional and atypical voltage protocols on DNA migration and separation are readily observed. Experiments illustrate that short periods of high voltage during electrophoretic injection do not destroy the quality of DNA separations, and in fact can enhance sample concentration five-fold. This study presents data that illustrate increases in average resolution, and resolution of longer fragments, obtained from electrophoretic injections utilizing voltages between 85 and 850 V/cm.