Recovery of control of posture and locomotion after a spinal cord injury: solutions staring us in the face.

Over the past 20 years, tremendous advances have been made in the field of spinal cord injury research. Yet, consumed with individual pieces of the puzzle, we have failed as a community to grasp the magnitude of the sum of our findings. Our current knowledge should allow us to improve the lives of patients suffering from spinal cord injury. Advances in multiple areas have provided tools for pursuing effective combination of strategies for recovering stepping and standing after a severe spinal cord injury. Muscle physiology research has provided insight into how to maintain functional muscle properties after a spinal cord injury. Understanding the role of the spinal networks in processing sensory information that is important for the generation of motor functions has focused research on developing treatments that sharpen the sensitivity of the locomotor circuitry and that carefully manage the presentation of proprioceptive and cutaneous stimuli to favor recovery. Pharmacological facilitation or inhibition of neurotransmitter systems, spinal cord stimulation, and rehabilitative motor training, which all function by modulating the physiological state of the spinal circuitry, have emerged as promising approaches. Early technological developments, such as robotic training systems and high-density electrode arrays for stimulating the spinal cord, can significantly enhance the precision and minimize the invasiveness of treatment after an injury. Strategies that seek out the complementary effects of combination treatments and that efficiently integrate relevant technical advances in bioengineering represent an untapped potential and are likely to have an immediate impact. Herein, we review key findings in each of these areas of research and present a unified vision for moving forward. Much work remains, but we already have the capability, and more importantly, the responsibility, to help spinal cord injury patients now.

Caged neuron MEA: a system for long-term investigation of cultured neural network connectivity.

Traditional techniques for investigating cultured neural networks, such as the patch clamp and multi-electrode array, are limited by: (1) the number of identified cells which can be simultaneously electrically contacted, (2) the length of time for which cells can be studied, and (3) the lack of one-to-one neuron-to-electrode specificity. Here, we present a new device - the caged neuron multi-electrode array - which overcomes these limitations. This micro-machined device consists of an array of neurocages which mechanically trap a neuron near an extracellular electrode. While the cell body is trapped, the axon and dendrites can freely grow into the surrounding area to form a network. The electrode is bi-directional, capable of both stimulating and recording action potentials. This system is non-invasive, so that all constituent neurons of a network can be studied over its lifetime with stable one-to-one neuron-to-electrode correspondence. Proof-of-concept experiments are described to illustrate that functional networks form in a neurochip system of 16 cages in a 4 x 4 array, and that suprathreshold connectivity can be fully mapped over several weeks. The neurochip opens a new domain in neurobiology for studying small cultured neural networks.

Synergistic antileukemic effects between ABT-869 and chemotherapy involve downregulation of cell cycle-regulated genes and c-Mos-mediated MAPK pathway.

Internal tandem duplications (ITDs) of fms-like tyrosine kinase 3 (FLT3) receptor play an important role in the pathogenesis of acute myeloid leukemia (AML) and represent an attractive therapeutic target. ABT-869 has demonstrated potent effects in AML cells with FLT3-ITDs. Here, we provide further evidence that ABT-869 treatment significantly downregulates cyclins D and E but increases the expression of p21 and p27. ABT-869 induces apoptosis through downregulation of Bcl-xL and upregulation of BAK, BID and BAD. We also evaluate the combinations of ABT-869 and chemotherapy. ABT-869 demonstrates significant sequence-dependent synergism with cytarabine and doxorubicin in cell lines and primary leukemia samples. The optimal combination was validated in MV4-11 xenografts. Low-density array analysis revealed the synergistic interaction involved in downregulation of cell cycle and mitogen-activated protein kinase pathway genes. CCND1 and c-Mos were the most significantly inhibited targets on both transcriptional and translational levels. Treatment with short hairpin RNAs targeting either CCND1 or c-Mos further sensitized MV4-11 cells to ABT-869. These findings suggest that specific pathway genes were further targeted by adding chemotherapy and support the rationale of combination therapy. Thus, a clinical trial using sequence-dependent combination therapy with ABT-869 in AML is warranted.

Electrolysis-based diaphragm actuators.

This work presents a new electrolysis-based microelectromechanical systems (MEMS) diaphragm actuator. Electrolysis is a technique for converting electrical energy to pneumatic energy. Theoretically electrolysis can achieve a strain of 136 000% and is capable of generating a pressure above 200 MPa. Electrolysis actuators require modest electrical power and produce minimal heat. Due to the large volume expansion obtained via electrolysis, small actuators can create a large force. Up to 100 µm of movement was achieved by a 3 mm diaphragm. The actuator operates at room temperature and has a latching and reversing capability.

The pattern and frequency of t(14;18) translocation and immunophenotype in Asian follicular lymphoma.

AIMS: Follicular lymphoma is frequently associated with t(14;18)(q32;q21) translocation. This study was undertaken to determine the pattern of Bcl-2, CD10 and Bcl-6 expression in relation to t(14;18) translocation in follicular lymphoma from a cohort of a multi-ethnic Asian population. METHODS AND RESULTS: Sixty-two cases of follicular lymphoma were retrieved for immunohistochemistry, and t(14;18) translocation analysis by polymerase chain reaction and fluorescent in-situ hybridization techniques. Bcl-2 expression was present in 74% of the cases. CD10 expression was also relatively low (61%), with decreasing frequency of expression in high-grade tumours. Bcl-6 protein was expressed in most of the tumours (88%) regardless of the tumour grade. The t(14;18) translocation was detected in 46 cases (74%) with an extremely high rate of t(14;18) translocation in ethnic Indian cases (100%). CONCLUSION: The frequency of t(14;18) translocation in this series of follicular lymphomas was higher when compared with previous Asian reports, but in accordance with European and North American findings. CD10 expression is strongly associated with a t(14;18) translocation event, but the overall CD10 expression was relatively low, possibly due to the high proportion of high-grade tumours in the series. t(14;18) translocation was not associated with Bcl-2 or Bcl-6 expression.

A test microchip for evaluation of hermetic packaging technology for biomedical prosthetic implants.

The development of a test chip that will be used to evaluate a hermetic and biocompatible package for the driving CMOS circuitry of a retinal prosthesis is described. The package design is estimated to be about 2 x 2 x 0.3 mm(3) and will be formed by conformal layers of parylene and a metal (e.g. titanium) as inner and outer protections, respectively. The test chip has been specifically designed for evaluation of the packaging technology. It consists of many blocks of analog and digital components as well as relative humidity and temperature sensors. The test chip has more probe points than a typical chip, allowing a more thorough evaluation of circuit behavior during the testing. This chip will first be coated in a layer of parylene C and soaked in heated isotonic saline for an extended period of time. Every block in the chip will then be tested for functionality using the surface probe points. The next step is to coat the surface of another test chip with parylene and a metal and repeat these soak tests. The results will then be analyzed and mean time-to-failure for the different samples will then be computed. Using the accelerated testing paradigm, these results will then be extrapolated to mean time-to-failure in the operating intraocular environment. Parylene test structures have already undergone an accelerated lifetime test and results have been analyzed.

Recording advances for neural prosthetics.

An important challenge for neural prosthetics research is to record from populations of neurons over long periods of time, ideally for the lifetime of the patient. Two new advances toward this goal are described, the use of local field potentials (LFPs) and autonomously positioned recording electrodes. LFPs are the composite extracellular potential field from several hundreds of neurons around the electrode tip. LFP recordings can be maintained for longer periods of time than single cell recordings. We find that similar information can be decoded from LFP and spike recordings, with better performance for state decodes with LFPs and, depending on the area, equivalent or slightly less than equivalent performance for signaling the direction of planned movements. Movable electrodes in microdrives can be adjusted in the tissue to optimize recordings, but their movements must be automated to be a practical benefit to patients. We have developed automation algorithms and a meso-scale autonomous electrode testbed, and demonstrated that this system can autonomously isolate and maintain the recorded signal quality of single cells in the cortex of awake, behaving monkeys. These two advances show promise for developing very long term recording for neural prosthetic applications.

Feasibility of T-cell receptor gamma (TCRgamma) gene rearrangement on formalin-fixed, paraffin-embedded tissues by PCR assays.

INTRODUCTION: T- and B-lymphocytes are involved in recognition of foreign antigen by the specificity of their surface T-cell receptor and immunoglobulin, generated by gene rearrangement. Each T- and B-lymphocyte carries unique rearranged TCR or immunoglobulin gene, which has been applied to detect clonal from non-clonal T- and B-cell proliferation. METHODS: Paraffin-embedded biopsy tissues of 85 T-, 24 B-cell non-Hodgkin's lymphomas (NHL) of various subtypes, and seven reactive lymphoid hyperplasia were retrieved from the archives for determining the feasibility of TCRgamma gene rearrangement analysis by PCR assays in our laboratory. DNA was extracted by Proteinase K digestion. The analyses were performed by five PCR assays, and analysed on polyacrylamide gel. RESULTS: Clonal TCRgamma gene rearrangement was demonstrated in 69/85 (81.2%) of the cases. Selective rearrangement of specific Vgamma segment was observed, especially in peripheral T-cell lymphoma-unspecified and nasal NK/T-cell lymphoma. Clonal TCRgamma rearranged band was also demonstrated in 4/24 (16.7%) and 2/7 (28.6%) of B-NHL and reactive lymphoid tissues respectively. CONCLUSION: PCR assays were able to demonstrate clonal TCRgamma gene rearrangement in a high proportion of T-NHL. However, the PCR results should be interpreted carefully. A neoplasm should only be considered as T-cell type if it does not express any B-cell marker because TCRgamma is not lineage specific as shown by the presence of clonal TCRgamma gene rearrangement in B-NHL. Hence, the results for TCR gene rearrangement should always be interpreted in conjunction with histology and immunophenotyping.

Model-based normalization for iterative 3D PET image reconstruction.

We describe a method for normalization in 3D PET for use with maximum a posteriori (MAP) or other iterative model-based image reconstruction methods. This approach is an extension of previous factored normalization methods in which we include separate factors for detector sensitivity, geometric response, block effects and deadtime. Since our MAP reconstruction approach already models some of the geometric factors in the forward projection, the normalization factors must be modified to account only for effects not already included in the model. We describe a maximum likelihood approach to joint estimation of the count-rate independent normalization factors, which we apply to data from a uniform cylindrical source. We then compute block-wise and block-profile deadtime correction factors using singles and coincidence data, respectively, from a multiframe cylindrical source. We have applied this method for reconstruction of data from the Concorde microPET P4 scanner. Quantitative evaluation of this method using well-counter measurements of activity in a multicompartment phantom compares favourably with normalization based directly on cylindrical source measurements.

Detector development for microPET II: a 1 microl resolution PET scanner for small animal imaging.

We are currently developing a small animal positron emission tomography (PET) scanner with a design goal of 1 microlitre (1 mm3) image resolution. The detectors consist of a 12 x 12 array of 1 x 1 x 10 mm lutetium oxyorthosilicate (LSO) scintillator crystals coupled to a 64-channel photomultiplier tube (PMT) via 5 cm long optical fibre bundles. The optical fibre connection allows a high detector packing fraction despite the dead space surrounding the active region of the PMT. Optical fibre bundles made from different types of glass were tested for light transmission, and also their effects on crystal identification and energy resolution, and compared to direct coupling of the LSO arrays to the PMTs. We also investigated the effects of extramural absorber (EMA) in the fibre bundles. Based on these results, fibre bundles manufactured from F2 glass were selected. We built three pairs of prototype detectors (directly coupled LSO array, fibre bundle without EMA and fibre bundle with EMA) and measured flood histograms, energy resolution, intrinsic spatial resolution and timing resolution. The results demonstrated an intrinsic spatial resolution (FWHM) of 1.12 mm (directly coupled), 1.23 mm (fibre bundle without EMA coupling) and 1.27 mm (fibre bundle with EMA coupling) using an approximately 500 microm diameter Na-22 point source. Using a 330 microm outer diameter steel needle line source filled with F-18, spatial resolution for the detector with the EMA optical fibre bundle improved to 1.05 mm. The respective timing and energy FWHM values were 1.96 ns, 21% (directly coupled), 2.20 ns, 23% (fibre bundle without EMA) and 2.99 ns, 30% (fibre bundle with EMA). The peak-to-valley ratio in the flood histograms was better with EMA (5:1) compared to the optical fibre bundle without EMA (2.5:1), due to the decreased optical cross-talk. In comparison to the detectors used in our current generation microPET scanner, these detectors substantially improve on the spatial resolution, preserve the timing resolution and provide adequate energy resolution for a modern high-resolution animal PET tomograph.

Silicon couplers for microfluidic applications.

Several types of silicon fluidic coupler have been designed, fabricated, and tested to facilitate external connections to MEMS (microelectromechanical systems) fluidic devices. By using both bulk micromachining and DRIE (deep reactive ion-etching) techniques, couplers of different geometry have been produced for use with any standard MEMS fluidic port. In addition, couplers are easily modified to accommodate any arbitrary fluidic port geometry. For ease of use, these couplers interface with PEEK (polyetheretherketone) and fused-silica capillary tubing, both of which are commonly used in HPLC (high-performance liquid chromatography) systems and are supported by a wide range of plumbing products. Coupler performance was evaluated and an operating range of at least 0-8,963 kPa (0-1,300 psig) is attainable.

ROC and LROC analyses of the effects of lesion contrast, size, and signal-to-noise ratio on detectability in PET images.

UNLABELLED: Image quality in PET is typically assessed using measures such as contrast recovery, noise variation, and signal-to-noise ratio (SNR). However, these criteria do not directly reflect performance in the clinical use of the images. Lesion detection is a critical task in the clinical interpretation of many PET studies. A receiver operating characteristic (ROC) study is an accepted method for quantitatively evaluating detection performance with respect to factors that influence image quality. ROC and localization ROC (LROC) analyses were conducted to investigate the effects of lesion contrast, SNR, and size on detectability of hot lesions in PET images. METHODS: A thorax phantom was imaged with spheres of 3 sizes simulating lesions (0.45, 1.0, and 1.9 mL). The relative activity in the lesions and the total number of counts acquired were each varied by factors of 2 to ascertain the effects of contrast and SNR, respectively. Measured attenuation correction and a standard reconstruction protocol were used. Three nuclear medicine physicians and 6 medical physicists participated as readers, rating each image and indicating the suspected lesion location. The area under the calculated ROC and LROC curves (Az and Az,LROC) were used as measures of detection performance. RESULTS: Detection performance was shown to increase from virtually random (Az approximately 0.5, Az,LROC approximately 0.2) to superior (Az > 0.9, Az,LROC > 0.9) as lesion contrast was increased by 50% and as lesion SNR was doubled. Detection performance was not seen to vary when comparison was made using image-based measures alone. CONCLUSION: This study quantitatively shows that moderate increases in the image-based measures of lesion contrast and SNR give a relatively large increase in the task-based measure of lesion detection as measured by ROC and LROC analyses. Thus, techniques that give modest increases in lesion contrast or SNR are expected to improve detection. Results will be useful in evaluating improvement in detection for various reconstruction, acquisition, and data analysis methods that enhance contrast or noise performance.

A micromachined chip-based electrospray source for mass spectrometry.

A micromachining process is described for fabricating a mass spectrometry electrospray source on a silicon chip. The process utilizes polymer (parylene) layers to form a system of chambers, filters, channels, and hollow needle structures (electrospray emitters) that extend more than a millimeter beyond the edge of the silicon substrate. The use of photoresist as the sacrificial layer facilitates the creation of long channels. Access to the channel structures on the chip is through a port etched through the silicon substrate that also serves as a sample reservoir. A reusable chip holder consisting of two plastic plates and an elastomer gasket provides the means to mount the chip in front of the mass spectrometer inlet and make electrical and gas connections. The electrospray emitters have tapered tips with 5 microns x 10 microns rectangular openings. The shape of the tip can be varied depending on the shape of the mask used to protect the parylene structures during the final plasma etch. The parylene emitters are physically robust and require only a high electric field to achieve stable electrospray operation over a period of a few hours. Direct comparisons with conventional glass or fused silica emitters indicated very similar performance with respect to signal strength and stability, spectral quality, and endurance. The automated MS/MS analysis of a mixture of tryptic peptides was no more difficult and yielded nearly identical results as the analysis of the same sample using a conventional nanospray device. This work demonstrates that an efficient electrospray interface to mass spectrometry can be integrated with other on-chip structures and mass-produced using a batch process.

Microstructures for studies of cultured neural networks.

A description is given of a functional silicon micromachined device that permits non-invasive, bidirectional, highly specific communication with cultured mammalian neurons. The heart of the system is a well structure that holds the cell in close proximity to a metal extracellular electrode while permitting normal outgrowth of axons and dendrites. An iterative approach is used to create a design that allows normal growth of the neurons while preventing their escape. An array of 16 such neurowells makes it possible to perform studies of biological neural network development and function with unprecedented detail.

The neurochip: a new multielectrode device for stimulating and recording from cultured neurons.

The neurochip is a silicon micromachined device upon which cultured mammalian neurons can be continuously and individually monitored and stimulated. The neurochip is based upon a 4 x 4 array of metal electrodes, each of which has a caged well structure designed to hold a single mature cell body while permitting normal outgrowth of neural processes. We demonstrate that this device is capable of maintaining cell survival, and that the electrodes can both record and stimulate electrical activity in individual cells with no crosstalk between channels.