Long-termin vivomonitoring of gliotic sheathing of ultrathin entropic coated brain microprobes with fiber-based optical coherence tomography.

Objective.Microfabricated neuroprosthetic devices have made possible important observations on neuron activity; however, long-term high-fidelity recording performance of these devices has yet to be realized. Tissue-device interactions appear to be a primary source of lost recording performance. The current state of the art for visualizing the tissue response surrounding brain implants in animals is immunohistochemistry + confocal microscopy, which is mainly performed after sacrificing the animal. Monitoring the tissue response as it develops could reveal important features of the response which may inform improvements in electrode design.Approach.Optical coherence tomography (OCT), an imaging technique commonly used in ophthalmology, has already been adapted for imaging of brain tissue. Here, we use OCT to achieve real-time,in vivomonitoring of the tissue response surrounding chronically implanted neural devices. The employed tissue-response-provoking implants are coated with a plasma-deposited nanofilm, which has been demonstrated as a biocompatible and anti-inflammatory interface for indwelling devices. We evaluate the method by comparing the OCT results to traditional histology qualitatively and quantitatively.Main results.The differences in OCT signal across the implantation period between the plasma group and the control reveal that the plasma-type coating of otherwise rigid brain probes (glass) only slightly improve the glial encapsulation in the brain parenchyma indicating that geometrical or mechanical influences are dominating the encapsulation process.Significance.Our approach can long-term monitor and compare the tissue-response to chronically-implanted neural probes with and withour plasma coating in living animal models. Our findings provide valuable insigh to the well acknowledged yet not solved challenge.

Axial localization with modulated-illumination extended-depth-of-field microscopy.

High-speed volumetric imaging represents a challenge in microscopy applications. We demonstrate a technique for acquiring volumetric images based on the extended depth of field microscopy with a fast focal scan and modulated illumination. By combining two frames with different illumination ramps, we can perform local depth ranging of the sample at speeds of up to half the camera frame rate. Our technique is light efficient, provides diffraction-limited resolution, enables axial localization that is largely independent of sample size, and can be operated with any standard widefield microscope based on fluorescence or darkfield contrast as a simple add-on. We demonstrate the accuracy of axial localization and applications of the technique to various dynamic extended samples, including in-vivo mouse brain.

High-throughput label-free flow cytometry based on matched-filter compressive imaging.

We present a fast label-free computational flow cytometer based on a strategy of compressive imaging. Scattered light from flowing objects is sub-divided into user-defined basis patterns by a deformable mirror and routed to different detectors associated with each pattern. The patterns can be optimized to be matched to the object features of interest, thus facilitating object identification and separation. Compared to conventional scanning flow cytometers, our technique provides increased information capacity without sacrificing flow velocity. Unique features of our matched-filter strategy are that it can simultaneously probe multiple objects throughout large fields of view with long depths of field. In our proof-of-concept demonstrations, we achieve throughputs of over 10,000 particles/s, working at flow velocities of over 1m/s.

Reduced cell attachment to poly(2-hydroxyethyl methacrylate)-coated ventricular catheters in vitro.

The majority of patients with hydrocephalus are dependent on ventriculoperitoneal shunts for diversion of excess cerebrospinal fluid. Unfortunately, these shunts are failure-prone and over half of all life-threatening pediatric failures are caused by obstruction of the ventricular catheter by the brain's resident immune cells, reactive microglia and astrocytes. Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used for biomedical implants. The extreme hydrophilicity of PHEMA confers resistance to protein fouling, making it a strong candidate coating for ventricular catheters. With the advent of initiated chemical vapor deposition (iCVD), a solvent-free coating technology that creates a polymer in thin film form on a substrate surface by introducing gaseous reactant species into a vacuum reactor, it is now possible to apply uniform polymer coatings on complex three-dimensional substrate surfaces. iCVD was utilized to coat commercially available ventricular catheters with PHEMA. The chemical structure was confirmed on catheter surfaces using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. PHEMA coating morphology was characterized by scanning electron microscopy. Testing PHEMA-coated catheters against uncoated clinical-grade catheters in an in vitro hydrocephalus catheter bioreactor containing co-cultured astrocytes and microglia revealed significant reductions in cell attachment to PHEMA-coated catheters at both 17-day and 6-week time points. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1268-1279, 2018.

Dual fluorescence-absorption deconvolution applied to extended-depth-of-field microscopy.

Fast imaging over large volumes can be obtained in a simple manner with extended-depth-of-field (EDOF) microscopy. A standard technique of Wiener deconvolution can correct for the blurring inherent in EDOF images. We compare Wiener deconvolution with an alternative, parameter-free technique based on the dual reconstruction of fluorescence and absorption layers in a sample. This alternative technique provides significantly enhanced reconstruction contrast owing to a quadratic positivity constraint that intrinsically favors sparse solutions. We demonstrate the advantages of this technique with mouse neuronal images acquired in vivo.

Extended depth-of-field microscopy with a high-speed deformable mirror.

We present a wide-field fluorescence microscopy add-on that provides a fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror with an update rate of 20 kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.

Toward a better understanding of the cellular basis for cerebrospinal fluid shunt obstruction: report on the construction of a bank of explanted hydrocephalus devices.

OBJECTIVE Shunt obstruction by cells and/or tissue is the most common cause of shunt failure. Ventricular catheter obstruction alone accounts for more than 50% of shunt failures in pediatric patients. The authors sought to systematically collect explanted ventricular catheters from the Seattle Children's Hospital with a focus on elucidating the cellular mechanisms underlying obstruction. METHODS In the operating room, explanted hardware was placed in 4% paraformaldehyde. Weekly, samples were transferred to buffer solution and stored at 4°C. After consent was obtained for their use, catheters were labeled using cell-specific markers for astrocytes (glial fibrillary acidic protein), microglia (ionized calcium-binding adapter molecule 1), and choroid plexus (transthyretin) in conjunction with a nuclear stain (Hoechst). Catheters were mounted in custom polycarbonate imaging chambers. Three-dimensional, multispectral, spinning-disk confocal microscopy was used to image catheter cerebrospinal fluid-intake holes (10× objective, 499.2-µm-thick z-stack, 2.4-µm step size, Olympus IX81 inverted microscope with motorized stage and charge-coupled device camera). Values are reported as the mean ± standard error of the mean and were compared using a 2-tailed Mann-Whitney U-test. Significance was defined at p < 0.05. RESULTS Thirty-six ventricular catheters have been imaged to date, resulting in the following observations: 1) Astrocytes and microglia are the dominant cell types bound directly to catheter surfaces; 2) cellular binding to catheters is ubiquitous even if no grossly visible tissue is apparent; and 3) immunohistochemical techniques are of limited utility when a catheter has been exposed to Bugbee wire electrocautery. Statistical analysis of 24 catheters was performed, after excluding 7 catheters exposed to Bugbee wire cautery, 3 that were poorly fixed, and 2 that demonstrated pronounced autofluorescence. This analysis revealed that catheters with a microglia-dominant cellular response tended to be implanted for shorter durations (24.7 ± 6.7 days) than those with an astrocyte-dominant response (1183 ± 642 days; p = 0.027). CONCLUSIONS Ventricular catheter occlusion remains a significant source of shunt morbidity in the pediatric population, and given their ability to intimately associate with catheter surfaces, astrocytes and microglia appear to be critical to this pathophysiology. Microglia tend to be the dominant cell type on catheters implanted for less than 2 months, while astrocytes tend to be the most prevalent cell type on catheters implanted for longer time courses and are noted to serve as an interface for the secondary attachment of ependymal cells and choroid plexus.

Fabrication of three-dimensional hydrogel scaffolds for modeling shunt failure by tissue obstruction in hydrocephalus.

BACKGROUND: Shunt obstruction in the treatment of hydrocephalus is poorly understood, is multi-factorial, and in many cases is modeled ineffectively. Several mechanisms may be responsible, one of which involves shunt infiltration by reactive cells from the brain parenchyma. This has not been modeled in culture and cannot be consistently examined in vivo without a large sample size. METHODS: We have developed and tested a three-dimensional in vitro model of astrocyte migration and proliferation around clinical grade ventricular catheters and into catheter holes that mimics the development of cellular outgrowth from the parenchyma that may contribute to shunt obstruction. RESULTS: Cell attachment and growth was observed on shunt catheters for as long as 80 days with at least 77% viability until 51 days. The model can be used to study cellular attachment to ventricular catheters under both static and pulsatile flow conditions, which better mimic physiological cerebrospinal fluid dynamics and shunt system flow rates (0.25 mL/min, 100 pulses/min). Pulsatile flow through the ventricular catheter decreased cell attachment/growth by 63% after 18 h. Under both conditions it was possible to observe cells accumulating around and in shunt catheter holes. CONCLUSIONS: Alone or in combination with previously-published culture models of shunt obstruction, this model serves as a relevant test bed to analyze mechanisms of shunt failure and to test catheter modifications that will prevent cell attachment and growth.

Quantitative 3-D analysis of GFAP labeled astrocytes from fluorescence confocal images.

BACKGROUND: There is a need for effective computational methods for quantifying the three-dimensional (3-D) spatial distribution, cellular arbor morphologies, and the morphological diversity of brain astrocytes to support quantitative studies of astrocytes in health, injury, and disease. NEW METHOD: Confocal fluorescence microscopy of multiplex-labeled (GFAP, DAPI) brain tissue is used to perform imaging of astrocytes in their tissue context. The proposed computational method identifies the astrocyte cell nuclei, and reconstructs their arbors using a local priority based parallel (LPP) tracing algorithm. Quantitative arbor measurements are extracted using Scorcioni's L-measure, and profiled by unsupervised harmonic co-clustering to reveal the morphological diversity. RESULTS: The proposed method identifies astrocyte nuclei, generates 3-D reconstructions of their arbors, and extracts quantitative arbor measurements, enabling a morphological grouping of the cell population. COMPARISON WITH EXISTING METHODS: Our method enables comprehensive spatial and morphological profiling of astrocyte populations in brain tissue for the first time, and overcomes limitations of prior methods. Visual proofreading of the results indicate a >95% accuracy in identifying astrocyte nuclei. The arbor reconstructions exhibited 3.2% fewer erroneous jumps in tracing, and 17.7% fewer false segments compared to the widely used fast-marching method that resulted in 9% jumps and 20.8% false segments. CONCLUSIONS: The proposed method can be used for large-scale quantitative studies of brain astrocyte distribution and morphology.

Population-scale three-dimensional reconstruction and quantitative profiling of microglia arbors.

MOTIVATION: The arbor morphologies of brain microglia are important indicators of cell activation. This article fills the need for accurate, robust, adaptive and scalable methods for reconstructing 3-D microglial arbors and quantitatively mapping microglia activation states over extended brain tissue regions. RESULTS: Thick rat brain sections (100-300 µm) were multiplex immunolabeled for IBA1 and Hoechst, and imaged by step-and-image confocal microscopy with automated 3-D image mosaicing, producing seamless images of extended brain regions (e.g. 5903 × 9874 × 229 voxels). An over-complete dictionary-based model was learned for the image-specific local structure of microglial processes. The microglial arbors were reconstructed seamlessly using an automated and scalable algorithm that exploits microglia-specific constraints. This method detected 80.1 and 92.8% more centered arbor points, and 53.5 and 55.5% fewer spurious points than existing vesselness and LoG-based methods, respectively, and the traces were 13.1 and 15.5% more accurate based on the DIADEM metric. The arbor morphologies were quantified using Scorcioni's L-measure. Coifman's harmonic co-clustering revealed four morphologically distinct classes that concord with known microglia activation patterns. This enabled us to map spatial distributions of microglial activation and cell abundances. AVAILABILITY AND IMPLEMENTATION: Experimental protocols, sample datasets, scalable open-source multi-threaded software implementation (C++, MATLAB) in the electronic supplement, and website (www.farsight-toolkit.org). http://www.farsight-toolkit.org/wiki/Population-scale_Three-dimensional_Reconstruction_and_Quanti-tative_Profiling_of_Microglia_Arbors CONTACT: broysam@central.uh.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quantitative arbor analytics: unsupervised harmonic co-clustering of populations of brain cell arbors based on L-measure.

This paper presents a robust unsupervised harmonic co-clustering method for profiling arbor morphologies for ensembles of reconstructed brain cells (e.g., neurons, microglia) based on quantitative measurements of the cellular arbors. Specifically, this method can identify groups and sub-groups of cells with similar arbor morphologies, and simultaneously identify the hierarchical grouping patterns among the quantitative arbor measurements. The robustness of the proposed algorithm derives from use of the diffusion distance measure for comparing multivariate data points, harmonic analysis theory, and a Haar-like wavelet basis for multivariate data smoothing. This algorithm is designed to be practically usable, and is embedded into the actively linked three-dimensional (3-D) visualization and analytics system in the free and open source FARSIGHT image analysis toolkit for interactive exploratory population-scale neuroanatomic studies. Studies on synthetic datasets demonstrate its superiority in clustering data matrices compared to recent hierarchical clustering algorithms. Studies on heterogeneous ensembles of real neuronal 3-D reconstructions drawn from the NeuroMorpho database show that the proposed method identifies meaningful grouping patterns among neurons based on arbor morphology, and revealing the underlying morphological differences.

Large-scale automated image analysis for computational profiling of brain tissue surrounding implanted neuroprosthetic devices using Python.

In this article, we describe the use of Python for large-scale automated server-based bio-image analysis in FARSIGHT, a free and open-source toolkit of image analysis methods for quantitative studies of complex and dynamic tissue microenvironments imaged by modern optical microscopes, including confocal, multi-spectral, multi-photon, and time-lapse systems. The core FARSIGHT modules for image segmentation, feature extraction, tracking, and machine learning are written in C++, leveraging widely used libraries including ITK, VTK, Boost, and Qt. For solving complex image analysis tasks, these modules must be combined into scripts using Python. As a concrete example, we consider the problem of analyzing 3-D multi-spectral images of brain tissue surrounding implanted neuroprosthetic devices, acquired using high-throughput multi-spectral spinning disk step-and-repeat confocal microscopy. The resulting images typically contain 5 fluorescent channels. Each channel consists of 6000 × 10,000 × 500 voxels with 16 bits/voxel, implying image sizes exceeding 250 GB. These images must be mosaicked, pre-processed to overcome imaging artifacts, and segmented to enable cellular-scale feature extraction. The features are used to identify cell types, and perform large-scale analysis for identifying spatial distributions of specific cell types relative to the device. Python was used to build a server-based script (Dell 910 PowerEdge servers with 4 sockets/server with 10 cores each, 2 threads per core and 1TB of RAM running on Red Hat Enterprise Linux linked to a RAID 5 SAN) capable of routinely handling image datasets at this scale and performing all these processing steps in a collaborative multi-user multi-platform environment. Our Python script enables efficient data storage and movement between computers and storage servers, logs all the processing steps, and performs full multi-threaded execution of all codes, including open and closed-source third party libraries.

Whole animal perfusion fixation for rodents.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate (5,7). Often, changes in response to hypoxia begin before the tissue can be preserved (12). The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures (3). It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Knockdown of cortical transthyretin expression around implanted neural prosthetic devices using intraventricular siRNA injection in the brain.

Neural prosthetic devices are showing increasing clinical use for the treatment of a variety of neurological disorders. However the functions on these devices are often limited due to an inability to effectively and chronically interface with neural tissue. The insertion of devices has been shown to result in significant cellular and vascular trauma surrounding the insertion site. In particular, the up-regulation of genes involved in neuronal degeneration are believed to contribute to the loss of neuronal tissue. RNA interference is a novel technique for the development of antisense therapeutics for the post-transcriptional silencing of specific genes. In order to demonstrate the feasibility of RNA interference for gene-specific silencing in vivo, a short interfering RNA targeting transthyretin, was infused prior to unilateral device insertion. Injection of siRNA was found to significantly reduce the expression of transthyretin mRNA when expression was assessed at 1 week following device insertion. Concomitant decreases in transthyretin protein levels were also observed. These data demonstrate the feasibility of using RNA interference to modulate the initial reactive cellular responses that occur in the brain following insertion of neural prosthetic devices.

The fate of ultrafast degrading polymeric implants in the brain.

We have recently reported on an ultrafast degrading tyrosine-derived terpolymer that degrades and resorbs within hours, and is suitable for use in cortical neural prosthetic applications. Here we further characterize this polymer, and describe a new tyrosine-derived fast degrading terpolymer in which the poly(ethylene glycol) (PEG) is replaced by poly(trimethylene carbonate) (PTMC). This PTMC containing terpolymer showed similar degradation characteristics but its resorption was negligible in the same period. Thus, changes in the polymer chemistry allowed for the development of two ultrafast degrading polymers with distinct difference in resorption properties. The in vivo tissue response to both polymers used as intraparenchymal cortical devices was compared to poly(lactic-co-glycolic acid) (PLGA). Slow resorbing, indwelling implant resulted in continuous glial activation and loss of neural tissue. In contrast, the fast degrading tyrosine-derived terpolymer that is also fast resorbing, significantly reduced both the glial response in the implantation site and the neuronal exclusion zone. Such polymers allow for brain tissue recovery, thus render them suitable for neural interfacing applications.

Ultrafast resorbing polymers for use as carriers for cortical neural probes.

We have identified a polymeric system based on a novel tyrosine-derived terpolymer that offers desirable insertion capability for flexible neural prosthetic devices. To test this concept, flexible films were coated with this terpolymer and their suitability for peranchyma insertion was visualized. The effect of the polymer on neural recording was evaluated using coated microwire probes. The stiff but readily resorbable polymer rapidly degrades (molecular weight half-life of 170 min) while turning into a soft gel, followed by complete resorption within 240 min. This polymeric platform maintains sufficient stiffness to facilitate pial penetration with a dry elastic modulus of 393±44 MPa but loses its strength within 30 min once immersed in saline. In vitro, the polymer's ability to locally deliver dexamethasone has been confirmed through a first order release profile over a 360 min period. In vitro, coated microwire probes regained their original impedance values of 0.5 KΩ within 20 min of wetting via water absorption and polymer resorption. In vivo, the retention of electrical recording capability was also demonstrated through multiple waveform detection in live animals. The ultrafast resorbing polymer as a platform to facilitate the implantation of micronized flexible probes can be utilized in future designs of chronic neural devices.

The effect of acoustically-induced cavitation on the permeance of a bullfrog urinary bladder.

It is well known that ultrasound enhances drug delivery to tissues, although there is not a general consensus about the responsible mechanisms. However, it is known that the most important factor associated with ultrasonically-enhanced drug permeance through tissues is cavitation. Here we report results from research conducted using a experimental approach adapted from single bubble sonoluminescence experiments which generates very well defined acoustic fields and allows controlled activation and location of cavitation. The experimental design requires that a biological tissue be immersed inside a highly degassed liquid media to avoid random bubble nucleation. Therefore, live frog bladders were used as the living tissue due to their high resistance to hypoxia. Tissue membrane permeance was measured using radiolabeled urea. The results show that an increase in tissue permeance only occurs when cavitation is present near the tissue membrane. Moreover, confocal microscopy shows a direct correlation between permeance increases and physical damage to the tissue.

Optical monitoring of neural networks evoked by focal electrical stimulation on microelectrode arrays using FM dyes.

Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 microm away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 microm) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 microm from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.

Robust adaptive 3-D segmentation of vessel laminae from fluorescence confocal microscope images and parallel GPU implementation.

This paper presents robust 3-D algorithms to segment vasculature that is imaged by labeling laminae, rather than the lumenal volume. The signal is weak, sparse, noisy, nonuniform, low-contrast, and exhibits gaps and spectral artifacts, so adaptive thresholding and Hessian filtering based methods are not effective. The structure deviates from a tubular geometry, so tracing algorithms are not effective. We propose a four step approach. The first step detects candidate voxels using a robust hypothesis test based on a model that assumes Poisson noise and locally planar geometry. The second step performs an adaptive region growth to extract weakly labeled and fine vessels while rejecting spectral artifacts. To enable interactive visualization and estimation of features such as statistical confidence, local curvature, local thickness, and local normal, we perform the third step. In the third step, we construct an accurate mesh representation using marching tetrahedra, volume-preserving smoothing, and adaptive decimation algorithms. To enable topological analysis and efficient validation, we describe a method to estimate vessel centerlines using a ray casting and vote accumulation algorithm which forms the final step of our algorithm. Our algorithm lends itself to parallel processing, and yielded an 8 x speedup on a graphics processor (GPU). On synthetic data, our meshes had average error per face (EPF) values of (0.1-1.6) voxels per mesh face for peak signal-to-noise ratios from (110-28 dB). Separately, the error from decimating the mesh to less than 1% of its original size, the EPF was less than 1 voxel/face. When validated on real datasets, the average recall and precision values were found to be 94.66% and 94.84%, respectively.

Effects of glial cells on electrode impedance recorded from neuralprosthetic devices in vitro.

Neural prosthetic devices hold the potential to be used in the treatment of a variety of neurological disorders. However, their long-term clinical success is currently limited by the ability to achieve stable interfaces between devices and the CNS. Immunohistochemical analysis has shown that cellular responses occur in tissue surrounding implanted devices. These cellular responses have been correlated with the impedance measured from device electrodes, leading to the hypothesis that a possible mechanism resulting in inconsistent device performance is the formation of an electrically insulating glial sheath at the implantation site. However, little is known about what cellular and tissue changes affect impedance values and thus contribute to the decreases in electrode performance. We have designed an in vitro system in which cell conditions can be varied within an artificial tissue matrix surrounding a neural prosthetic device. In this study, high-density cultures of glial cells were analyzed by immunohistochemical methods and impedance spectroscopy. Astrocytes and microglia were cultured at various ratios within the matrix surrounding the probes, and were observed over a period of 2 weeks. Cell seeding conditions and confocal images were compared to impedance data to enable the effects of glial cell type on electrode impedance to be determined.