Biomedical subjects

J N Turner

Publications and source records attributed to J N Turner.

At least 19 recordsLinked to original sources

PubMed2006

Effects of insertion conditions on tissue strain and vascular damage during neuroprosthetic device insertion.

Long-term integration of neuroprosthetic devices is challenged by reactive responses that compromise the brain-device interface. The contribution of physical insertion parameters to immediate damage is not well described. We have developed an ex vivo preparation to capture real-time images of tissue deformation during device insertion using thick tissue slices from rat brains prepared with fluorescently labeled vasculature. Qualitative and quantitative assessments of damage were made for insertions using devices with different tip shapes inserted at different speeds. Direct damage to the vasculature included severing, rupturing and dragging, and was often observed several hundred micrometers from the insertion site. Slower insertions generally resulted in more vascular damage. Cortical surface features greatly affected insertion success; insertions attempted through pial blood vessels resulted in severe tissue compression. Automated image analysis techniques were developed to quantify tissue deformation and calculate mean effective strain. Quantitative measures demonstrated that, within the range of experimental conditions studied, faster insertion of sharp devices resulted in lower mean effective strain. Variability within each insertion condition indicates that multiple biological factors may influence insertion success. Multiple biological factors may contribute to tissue distortion, thus a wide variability was observed among insertions made under the same conditions.

Animals↗

PubMed2005

Dexamethasone treatment reduces astroglia responses to inserted neuroprosthetic devices in rat neocortex.

Microfabricated neural prosthetic devices hold great potential for increasing knowledge of brain function and treating patients with lost CNS function. Time-dependent loss of brain-device communication limits long-term use of these devices. Lost CNS function is associated with reactive responses that produce an encapsulating cellular sheath. Since early reactive responses may be associated with injuries produced at the time of device insertion, for example, vascular damage and disruption of the blood-brain barrier, we tested the effectiveness of the synthetic glucocorticoid, dexamethasone, in controlling insertion- and device-associated reactive responses. Dexamethasone (200 microg/kg) was administered as subcutaneous injections for 1 or 6 days beginning on the day of device insertion. Single shank microfabricated silicon devices were inserted into pre-motor cortex of adult rats. Reactive responses were assessed by immunohistochemistry for glial fibrillary acidic protein (astrocytes), CD11b (microglia), and laminin that labeled extracellular protein deposited around the insertion site and in association with vascular elements. Data were collected by confocal microscopy imaging of 100-microm-thick tissue slices. Reactive responses in vehicle control animals were similar to non-injected control animals. Dexamethasone treatment profoundly effected early and sustained reactive responses observed 1 and 6 weeks following device insertion, respectively. Dexamethasone treatment greatly attenuated astroglia responses, while microglia and vascular responses appeared to be increased. The 6-day treatment was more effective than the single injection regime. These results suggest that anti-inflammatory agents can be used to control reactive responses around inserted neural prosthetic devices and may provide a means to insure their long-term function.

Animals↗

PubMed2004

Topographically modified surfaces affect orientation and growth of hippocampal neurons.

Extracellular matrix molecules provide biochemical and topographical cues that influence cell growth in vivo and in vitro. Effects of topographical cues on hippocampal neuron growth were examined after 14 days in vitro. Neurons from hippocampi of rat embryos were grown on poly-L-lysine-coated silicon surfaces containing fields of pillars with varying geometries. Photolithography was used to fabricate 1 microm high pillar arrays with different widths and spacings. Beta(III)-tubulin and MAP-2 immunocytochemistry and scanning electron microscopy were used to describe neuronal processes. Automated two-dimensional tracing software quantified process orientation and length. Process growth on smooth surfaces was random, while growth on pillared surfaces exhibited the most faithful alignment to pillar geometries with smallest gap sizes. Neurite lengths were significantly longer on pillars with the smallest inter-pillar spacings (gaps) and 2 microm pillar widths. These data indicate that physical cues affect neuron growth, suggesting that extracellular matrix topography may contribute to cell growth and differentiation. These results demonstrate new strategies for directing and promoting neuronal growth that will facilitate studies of synapse formation and function and provide methods to establish defined neural networks.

Animals↗

PubMed2003

Brain responses to micro-machined silicon devices.

Micro-machined neural prosthetic devices can be designed and fabricated to permit recording and stimulation of specific sites in the nervous system. Unfortunately, the long-term use of these devices is compromised by cellular encapsulation. The goals of this study were to determine if device size, surface characteristics, or insertion method affected this response. Devices with two general designs were used. One group had chisel-shaped tips, sharp angular corners, and surface irregularities on the micrometer size scale. The second group had rounded corners, and smooth surfaces. Devices of the first group were inserted using a microprocessor-controlled inserter. Devices of the second group were inserted by hand. Comparisons were made of responses to the larger devices in the first group with devices from the second group. Responses were assessed 1 day and 1, 2, 4, 6, and 12 weeks after insertions. Tissues were immunochemically labeled for glial fibrillary acidic protein (GFAP) or vimentin to identify astrocytes, or for ED1 to identify microglia. For the second comparison devices from the first group with different cross-sectional areas were analyzed. Similar reactive responses were observed following insertion of all devices; however, the volume of tissue involved at early times, <1 week, was proportional to the cross-sectional area of the devices. Responses observed after 4 weeks were similar for all devices. Thus, the continued presence of devices promotes formation of a sheath composed partly of reactive astrocytes and microglia. Both GFAP-positive and -negative cells were adherent to all devices. These data indicate that device insertion promotes two responses-an early response that is proportional to device size and a sustained response that is independent of device size, geometry, and surface roughness. The early response may be associated with the amount of damage generated during insertion. The sustained response is more likely due to tissue-device interactions.

Animals↗

PubMed2003

Attenuation correction in confocal laser microscopes: a novel two-view approach.

Confocal microscopy is a three-dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth-dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal-to-noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two-view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180 degrees with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model-based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space-filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth-dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two-view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.

Animals↗

PubMed2003

Algorithms for accurate 3D registration of neuronal images acquired by confocal scanning laser microscopy.

This paper presents automated and accurate algorithms based on high-order transformation models for registering three-dimensional (3D) confocal images of dye-injected neurons. The algorithms improve upon prior methods in several ways, and meet the more stringent image registration needs of applications such as two-view attenuation correction recently developed by us. First, they achieve high accuracy ( approximately 1.2 voxels, equivalent to 0.4 micro m) by using landmarks, rather than intensity correlations, and by using a high-dimensional affine and quadratic transformation model that accounts for 3D translation, rotation, non-isotropic scaling, modest curvature of field, distortions and mechanical inconsistencies introduced by the imaging system. Second, they use a hierarchy of models and iterative algorithms to eliminate potential instabilities. Third, they incorporate robust statistical methods to achieve accurate registration in the face of inaccurate and missing landmarks. Fourth, they are fully automated, even estimating the initial registration from the extracted landmarks. Finally, they are computationally efficient, taking less than a minute on a 900-MHz Pentium III computer for registering two images roughly 70 MB in size. The registration errors represent a combination of modelling, estimation, discretization and neuron tracing errors. Accurate 3D montaging is described; the algorithms have broader applicability to images of vasculature, and other structures with distinctive point, line and surface landmarks.

Algorithms↗

PubMed2002

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.

Amino Acid Sequence↗

PubMed2001

Axonal outgrowth of hippocampal neurons on micro-scale networks of polylysine-conjugated laminin.

Microcontact printing was used to define an interconnected lattice network of polylysine-conjugated laminin, a protein-polypeptide ligate that is an effective promoter of neuron outgrowth on material surfaces. In the presence of serum proteins, rat hippocampal neurons selectively adhered to features of polylysine-conjugated laminin as narrow as 2.6 microm in width. Adhering neurons extended long axonal processes, which precisely followed and did not deviate from the prescribed patterns, demonstrating that neurons respond to this protein with high selectivity and that these techniques effectively provide long-range guidance of axonal outgrowth. Further examination of neuron response under serum-free cell culture conditions demonstrated that the outgrowth-promoting activity of polylysine-conjugated laminin was attributed to biologically active laminin. Together, these results demonstrate that polylysine-conjugated laminin provides for high-precision guidance of neuron attachment and axon outgrowth on material surfaces in a serum-independent manner. This ability to guide hippocampal neuron response in low-density, serum-free culture with high precision is valuable for the development of advanced, neuron-based devices.

Animals↗

PubMed2001

Optimal scheduling of tracing computations for real-time vascular landmark extraction from retinal fundus images.

Recently, this group published fast algorithms for automatic tracing (vectorization) of the vasculature in live retinal angiograms, and for the extraction of visual landmarks formed by vascular bifurcations and crossings. These landmarks are used for feature-based image matching for controlling a computer-assisted laser retinal surgery instrument currently under development. This paper describes methods to schedule the vascular tracing computations to maximize the rate of growth of quality of the partial tracing results within a frame cycle. There are two main advantages. First, progressive image matching from partially extracted landmark sets can be faster, and provide an earlier indication of matching failure. Second, the likelihood of successful image matching is greatly improved since the extracted landmarks are of the highest quality for the given computational budget. The scheduling method is based on quantitative measures for the computational work and the quality of landmarks. A coarse grid-based analysis of the image is used to generate seed points for the tracing computations, along with estimates of local edge strengths, orientations, and vessel thickness. These estimates are used to define criteria for real-time preemptive scheduling of the tracing computations. It is shown that the optimal schedule can only be achieved in perfect hindsight, and is thus unrealizable. This leads to scheduling heuristics that approximate the behavior of the optimal algorithm. One such approximation produced approximately 400% improvement in the quality of the partial results at a defined milestone, as compared to random scheduling. The resulting algorithm can be readily implemented on conventional and multiple-processor systems, and is being applied to computer-assisted laser retinal surgery.

Algorithms↗

PubMed2000

Attachment of astroglial cells to microfabricated pillar arrays of different geometries.

We studied the attachment of astroglial cells on smooth silicon and arrays of silicon pillars and wells with various widths and separations. Standard semiconductor industry photolithographic techniques were used to fabricate pillar arrays and wells in single-crystal silicon. The resulting pillars varied in width from 0. 5 to 2.0 micrometer, had interpillar gaps of 1.0-5.0 micrometer, and were 1.0 micrometer in height. Arrays also contained 1.0-micromter-deep wells that were 0.5 micrometer in diameter and separated by 0.5-2.0 micrometer. Fluorescence, reflectance, and confocal light microscopies as well as scanning electron microscopy were used to quantify cell attachment, describe cell morphologies, and study the distribution of cytoskeletal proteins actin and vinculin on surfaces with pillars, wells, and smooth silicon. Seventy percent of LRM55 astroglial cells displayed a preference for pillars over smooth silicon, whereas only 40% preferred the wells to the smooth surfaces. Analysis of variance statistics performed on the data sets yielded values of p > approximately.5 for the comparison between pillar data sets and < approximately.0003 in the comparison between pillar and well data sets. Actin and vinculin distributions were highly polarized in cells found on pillar arrays. Scanning electron microscopy clearly demonstrated that cells made contact with the tops of the pillars and did not reach down into the spaces between pillars even when the interpillar gap was 5.0 microm. These experiments support the use of surface topography to direct the attachment, growth, and morphology of cells. These surfaces can be used to study fundamental cell properties such as cell attachment, proliferation, and gene expression. Such topography might also be used to modify implantable medical devices such as neural implants and lead to future developments in tissue engineering.

Actins↗

PubMed1999

In vivo activation and in situ BDNF-stimulated nuclear translocation of mitogen-activated/extracellular signal-regulated protein kinase is inhibited by ethanol in the developing rat hippocampus.

In order to test the hypothesis that ethanol (EtOH)-induced changes in growth factor signal transduction contribute to the teratogenic effects of EtOH in the developing brain, neonatal rat pups were administered a single dose of EtOH during the brain growth spurt (5 days of age, PN5). Hippocampal mitogen-activated/extracellular signal-regulated protein kinase (MAPK/ERK) activation was analyzed one to 6 h after exposure by electrophoretic-mobility shift assay combined with western blot. Brain-Derived Neurotrophic Factor (BDNF) was used to stimulate ERK in hippocampal slices prepared from PN5 pups and activation and cellular localization was determined with immunofluorescence combined with confocal microscopy. EtOH decreased ERK activation in vivo and decreased nuclear translocation of BDNF-stimulated ERK in situ. These data suggest EtOH-induced inhibition of growth factor signaling may contribute to the development of fetal alcohol syndrome and alcohol-related birth defects.

Animals↗

PubMed1999

Cerebral astrocyte response to micromachined silicon implants.

The treatment of neurologic disorders and the restoration of lost function due to trauma by neuroprosthetic devices has been pursued for over 20 years. The methodology for fabricating miniature devices with sophisticated electronic functions to interface with nervous system tissue is available, having been well established by the integrated circuit industry. Unfortunately, the effectiveness of these devices is severely limited by the tissue reaction to the insertion and continuous presence of the implant, a foreign object. This study was designed to document the response of reactive astrocytes in the hope that this information will be useful in specifying new fabrication technologies and devices capable of prolonged functioning in the brain. Model probes fabricated from single crystal silicon wafers were implanted into the cerebral cortices of rats. The probes had a 1 x 1-mm tab, for handling, and a 2-mm-long shaft with a trapezoidal cross-section (200-microm base, 60microm width at the top, and 130 microm height). The tissue response was studied by light and scanning electron microscopy at postinsertion times ranging from 2 to 12 weeks. A continuous sheath of cells was found to surround the insertion site in all tissue studied and was well developed but loosely organized at 2 weeks. By 6 and 12 weeks, the sheath was highly compacted and continuous, isolating the probe from the brain. At 2 and 4 weeks, the sheath was disrupted when the probe was removed from the fixed tissue, indicating that cells attached more strongly to the surface of the probe than to the nearby tissue. The later times showed much less disruption. Scanning electron microscopy of the probes showed adherent cells or cell fragments at all time points. Thus, as the sheath became compact, the cells on the probe and the cells in the sheath had decreased adhesion to each other. Immunocytochemistry demonstrated that the sheath was labeled with antibodies to glial fibrillary acidic protein (GFAP), an indicator for reactive gliosis. The tissue surrounding the insertion site showed an increased number of GFAP-positive cells which tended to return to control levels as a function of time after probe insertion. It was concluded that reactive gliosis is an important part of the process forming the cellular sheath. Further, the continuous presence of the probe appears to result in a sustained response that produces and maintains a compact sheath, at least partially composed of reactive glia, which isolates the probe from the brain.

Animals↗

PubMed1999

Correlation of astroglial cell function on micro-patterned surfaces with specific geometric parameters.

Microcontact printing techniques were used to modify silicon substrates with arrays of hexagonal features of N1[3-(trimethoxysilyl) propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS), which are hydrophilic, cell-adhesive and hydrophobic, non-adhesive organosilanes, respectively. In the presence of serum proteins, LRM55 cell adhesion and morphology on these modified surfaces were best correlated to the width of the cell-adhesive features. On surfaces modified with small (5 microm in width) cell-adhesive features, LRM55 cells elaborated only thin processes. As feature width was increased, cells on these surfaces exhibited increased cell spreading and elaborated wide processes. On surfaces modified with large (>35 microm in width) features, single cells adhered to and spread upon individual DETA features. In a similar fashion, LRM55 cell adhesion density increased with increasing feature width; this correlation could be represented by a simple, second-order relation, and was independent of all other measures of pattern geometry. The results of this study provide evidence that micro-patterning may be effective in controlling astrocyte interaction with implant materials.

Animals↗

PubMed1999

Rapid automated tracing and feature extraction from retinal fundus images using direct exploratory algorithms.

Algorithms are presented for rapid, automatic, robust, adaptive, and accurate tracing of retinal vasculature and analysis of intersections and crossovers. This method improves upon prior work in several ways: 1) automatic adaptation from frame to frame without manual initialization/adjustment, with few tunable parameters; 2) robust operation on image sequences exhibiting natural variability, poor and varying imaging conditions, including over/under-exposure, low contrast, and artifacts such as glare; 3) does not require the vasculature to be connected, so it can handle partial views; and 4) operation is efficient enough for use on unspecialized hardware, and amenable to deadline-driven computing, being able to produce a rapidly and monotonically improving sequence of usable partial results. Increased computation can be traded for superior tracing performance. Its efficiency comes from direct processing on gray-level data without any preprocessing, and from processing only a minimally necessary fraction of pixels in an exploratory manner, avoiding low-level image-wide operations such as thresholding, edge detection, and morphological processing. These properties make the algorithm suited to real-time, on-line (live) processing and is being applied to computer-assisted laser retinal surgery.

Algorithms↗

PubMed1998

Three-dimensional changes of the cytoskeleton of vascular endothelial cells exposed to sustained hydrostatic pressure.

The three-dimensional changes in the cytoskeleton and in cell proliferation of bovine pulmonary artery endothelial cells when exposed to sustained hydrostatic pressure were investigated in vitro using laser scanning confocal microscopy. Subconfluent endothelial cells on rigid substrates were exposed to 1.5, 5 and 10 cm H2O pressure under hydrostatic heads of culture medium for up to seven days. Confocal microscopic images were taken at distances of 0.4 micron through the thickness of the sample and visualised in multiplanar, stereopair and 90 degrees rotation formats. The results of the study provide evidence of: increased proliferation after exposure to 10 cm H2O pressure for five and seven days; cell bilayering after exposure to 1.5 and 5 cm H2O pressure and trilayering after exposure to 10 cm H2O pressure for seven days; and F-actin filament reorganisation into centrally located, parallel, stress fibres in confluent cells, into peripheral bands in subconfluent, multilayered cells, and into multilayers in the plane perpendicular to the applied force.

Animals↗

PubMed1998

Image processing algorithms for retinal montage synthesis, mapping, and real-time location determination.

Although laser retinal surgery is the best available treatment for choridal neovascularization, the current procedure has a low success rate (50%). Challenges, such as motion-compensated beam steering, ensuring complete coverage and minimizing incidental photodamage, can be overcome with improved instrumentation. This paper presents core image processing algorithms for 1) rapid identification of branching and crossover points of the retinal vasculature; 2) automatic montaging of video retinal angiograms; 3) real-time location determination and tracking using a combination of feature-tagged point-matching and dynamic-pixel templates. These algorithms tradeoff conflicting needs for accuracy, robustness to image variations (due to movements and the difficulty of providing steady illumination) and noise, and operational speed in the context of available hardware. The algorithm for locating vasculature landmarks performed robustly at a speed of 16-30 video image frames/s depending upon the field on a Silicon Graphics workstation. The montaging algorithm performed at a speed of 1.6-4 s for merging 5-12 frames. The tracking algorithm was validated by manually locating six landmark points on an image sequence with 180 frames, demonstrating a mean-squared error of 1.35 pixels. It successfully detected and rejected instances when the image dimmed, faded, lost contrast, or lost focus.

Algorithms↗

PubMed1998

Accuracy of nuclear classification in cervical smear images. Quantitative impact of computational deconvolution and 3-D feature computation.

OBJECTIVE: To investigate the accuracy with which the nuclei of cells in overlapped and thick clusters in cervical/ vaginal smears can be classified independent of the segmentation algorithm used and to determine the influence of three-dimensional (3-D) processing as compared to two-dimensional (2-D) methods on classification of the nuclei. STUDY DESIGN: Cell clusters were imaged from 31 ThinPrep smears composed of 808 nuclei, of which 420 were determined to be abnormal by a cytotechnologist. Sets of 2-D and 3-D volumetric features of the detected nuclei were formulated, and classifiers were constructed. The effect of computational deconvolution on classification was assessed using nearest-neighbor and Wiener filter in 2-D and 3-D before calculating features. A "best focus plane" was calculated for each nucleus from the 3-D data set, and the 2-D features in this plane were also analyzed.

Algorithms↗

PubMed1998

Advances in high-speed, three-dimensional imaging and automated segmentation algorithms for thick and overlapped clusters in cytologic preparations. Application to cervical smears.

OBJECTIVE: To use three-dimensional (3-D) imaging and localized adaptive image analysis to enable automated cervical smear screening systems to efficiently and effectively process thick and overlapped cell clusters currently left unprocessed. STUDY DESIGN: Instrumentation was developed to perform high-speed (50-200 optical sections per second at 256 x 256 resolution), 3-D imaging of thick regions of cervical smears. Normal and abnormal ThinPrep smears were imaged at two levels of resolution to approximate higher-resolution, wide-area imaging. Improved dual-resolution, 3-D image analysis algorithms were developed for segmenting nuclei in these clusters. RESULTS: Despite low contrast, high variability and dense overlaps, the algorithms detected 89% and correctly segmented 76% of nuclei in clusters from normal smears and detected 75% and correctly segmented 45% of nuclei in clusters from abnormal smears in low-resolution images. In high-resolution images they detected 88% and segmented 76% of nuclei from normal specimens and detected 55% and segmented 45% of nuclei from abnormal specimens. At least one nucleus from each cell cluster was correctly segmented. CONCLUSION: Selective application of 3-D imaging and 3-D image analysis to thick and overlapped regions can enable a significant fraction (45-89%) of clustered and embedded cells to be accessed by an automated analysis system. These regions are, for the most part, unprocessable by current two-dimensional methods.

Algorithms↗