Advanced age is not a barrier to chronic intracortical single-unit recording in rat cortex.

Introduction: Available evidence suggests that as we age, our brain and immune system undergo changes that increase our susceptibility to injury, inflammation, and neurodegeneration. Since a significant portion of the potential patients treated with a microelectrode-based implant may be older, it is important to understand the recording performance of such devices in an aged population. Methods: We studied the chronic recording performance and the foreign body response (FBR) to a clinically used microelectrode array implanted in the cortex of 18-month-old Sprague Dawley rats. Results and discussion: To the best of our knowledge, this is the first preclinical study of its type in the older mammalian brain. Here, we show that single-unit recording performance was initially robust then gradually declined over a 12-week period, similar to what has been previously reported using younger adult rats and in clinical trials. In addition, we show that FBR biomarker distribution was similar to what has been previously described for younger adult rats implanted with multi-shank recording arrays in the motor cortex. Using a quantitative immunohistochemcal approach, we observed that the extent of astrogliosis and tissue loss near the recording zone was inversely related to recording performance. A comparison of recording performance with a younger cohort supports the notion that aging, in and of itself, is not a limiting factor for the clinical use of penetrating microelectrode recording arrays for the treatment of certain CNS disorders.

Achieving biocompatibility and tailoring mechanical properties of SLA 3D printed devices for microfluidic and cell culture applications.

Stereolithography (SLA) and other photopolymerization-based additive manufacturing approaches are becoming popular for the fabrication of microfluidic devices and cell-infused platforms, but many of the resins employed in these techniques are cytotoxic to cells or do not have the appropriate mechanical properties for microfluidic components. Here, using a commercially available resin, we demonstrate that biocompatibility and a range of mechanical properties can be achieved through post-print optimization involving baking, soaking, network swelling, and UV exposure. We show that UV-vis spectrophotometry can be used to detect methacrylate monomer/oligomer, and utilizing this method, we found that baking at 120 °C for 24 hours was the optimal method for removing cytotoxic chemical species and creating nontoxic cell culture platforms, though UV exposure and soaking in 100% ethanol also can substantially reduce cytotoxicity. Furthermore, we show that the mechanical properties can be modified, including up to 50% for the Young's modulus and an order of magnitude for the flexural modulus, through the post-processing approach employed. Based on the study results, users can choose post-processing approaches to achieve needed cytotoxicity and mechanical profiles, simultaneously.

The assessment of adeno-associated vectors as potential intrinsic treatments for brainstem axon regeneration.

BACKGROUND: Adeno-associated virus (AAV) vector-mediated transgene expression is a promising therapeutic to change the intrinsic state of neurons and promote repair after central nervous system injury. Given that numerous transgenes have been identified as potential candidates, the present study demonstrates how to determine whether their expression by AAV has a direct intrinsic effect on axon regeneration. METHODS: Serotype 2 AAV-enhanced green fluorescent protein (EGFP) was stereotaxically injected into the brainstem of adult rats, followed by a complete transection of the thoracic spinal cord and Schwann cell (SC) bridge implantation. RESULTS: The expression of EGFP in brainstem neurons labeled numerous axons in the thoracic spinal cord and that regenerated into the SC bridge. The number of EGFP-labeled axons rostral to the bridge directly correlated with the number of EGFP-labeled axons that regenerated into the bridge. Animals with a greater number of EGFP-labeled axons rostral to the bridge exhibited an increased percentage of those axons found near the distal end of the bridge compared to animals with a lesser number. This suggested that EGFP may accumulate distally in the axon with time, enabling easier visualization. By labeling brainstem axons with EGFP before injury, numerous axon remnants undergoing Wallerian degeneration may be identified distal to the complete transection up to 6 weeks after injury. CONCLUSIONS: Serotype 2 AAV-EGFP enabled easy visualization of brainstem axon regeneration. Rigorous models of axonal injury (i.e. complete transection and cell implantation) should be used in combination with AAV-EGFP to directly assess AAV-mediated expression of therapeutic transgenes as intrinsic treatments to improve axonal regeneration.

Conductive Polymer Enabled Biostable Liquid Metal Electrodes for Bioelectronic Applications.

Gallium (Ga)-based liquid metal materials have emerged as a promising material platform for soft bioelectronics. Unfortunately, Ga has limited biostability and electrochemical performance under physiological conditions, which can hinder the implementation of its use in bioelectronic devices. Here, an effective conductive polymer deposition strategy on the liquid metal surface to improve the biostability and electrochemical performance of Ga-based liquid metals for use under physiological conditions is demonstrated. The conductive polymer [poly(3,4-ethylene dioxythiophene):tetrafluoroborate]-modified liquid metal surface significantly outperforms the liquid metal.based electrode in mechanical, biological, and electrochemical studies. In vivo action potential recordings in behaving nonhuman primate and invertebrate models demonstrate the feasibility of using liquid metal electrodes for high-performance neural recording applications. This is the first demonstration of single-unit neural recording using Ga-based liquid metal bioelectronic devices to date. The results determine that the electrochemical deposition of conductive polymer over liquid metal can improve the material properties of liquid metal electrodes for use under physiological conditions and open numerous design opportunities for next-generation liquid metal-based bioelectronics.

The challenge of integrating devices into the central nervous system.

Implanted biomedical devices are playing an increasingly important role in the treatment of central nervous system disorders. While devices such as deep brain stimulation electrodes and drug delivery systems have shown clinical success in chronic applications, other devices such as nerve guidance substrates and recording electrodes that operate over a very short length scale have not had the same kind of clinical impact. By reviewing what is currently known about the brain tissue response to implanted electrodes, the authors propose that the foreign-body response, which changes the tissue structure immediately surrounding implanted devices, may be the reason near-function devices are stalled in preclinical development. The article concludes by reviewing recent efforts to reduce the foreign body response, which shows promise to accelerate the clinical development of this new generation of biomedical devices.

Characterization of microglial attachment and cytokine release on biomaterials of differing surface chemistry.

The clinical usefulness of central nervous system recording electrodes is currently limited by inconsistent long-term performance that is believed to be governed by the brain tissue response to the implant. In this study, we observed persistent macrophage biomarker expression at the biotic-abiotic interface surrounding implanted electrodes over a 12-week indwelling period. Using the cell type-specific marker CD11b to examine the cells attached to electrodes retrieved over the indwelling period, we found that most of the cells were activated microglia, the resident macrophage of brain tissue, indicating that the implanted electrodes behave as a persistent inflammatory stimulus. To determine the potential usefulness of different materials as coatings for implanted electrodes, we examined brain-derived microglial cell attachment and cytokine release on a number of medically relevant materials. Our results suggest that activated microglia attach to many of the materials used as external coatings for electrode manufacture, and likely serve as a source of pro-inflammatory and neurotoxic cytokines that may be responsible for reducing the biocompatibility of such implants. Our results also indicate that low protein-binding coatings may be useful in reducing microglial attachment upon implantation in brain tissue and may provide a means of improving electrode biocompatibility.

The foreign body response and morphometric changes associated with mesh-style peripheral nerve cuffs.

Nerve cuffs have been used to anchor and protect penetrating electrodes in peripheral nerves and have been used as non-penetrating electrodes for neural recording and nerve stimulation. The material of choice for such applications is silicone, an inert synthetic biomaterial which elicits a minimal chronic foreign body response (FBR). While histological studies of solid silicone cuffs are available, to the best of our knowledge a comparison to other cuff designs is not well documented. Here, we describe the FBR and morphological changes that accompany nerve cuff implantation in the rat sciatic nerve by comparing a metallic mesh with and without a parylene coating to one made of silicone. Two months after implantation, we observed that such implants, irrespective of the cuff type, were associated with a persistent inflammatory response consisting of activated macrophages attached to the implant surfaces, which extended into the endoneurial space of the encapsulated nerve. We also observed foreign body giant cells in the epineurial space that were more prevalent in the mesh cohorts. The mesh cuff groups showed significant changes in several morphometric parameters that were not seen in the silicon group including reductions in nerve fiber packing density and a greater reduction of large diameter fibers. High magnification microscopy also showed greater evidence of foamy macrophages in the endoneurial space of the mesh implanted cohorts. Although the precise mechanisms are unknown, the results showed that mesh style nerve cuffs show a greater inflammatory response and had greater reductions in morphometric changes in the underlying nerve compared to silicone in the absence of a penetrating injury. STATEMENT OF SIGNIFICANCE: While traditional silicone cuffs have been in use for decades, the inflammatory and morphometric effects of these cuffs on the underlying nerve have not been deeply studied. Further, manipulation of the foreign body response to nerve cuffs by using various materials and/or designs has not been well reported. Therefore, we report the inflammatory response around nerve cuffs of various materials and designs, as well as report morphometric parameters of the underlying nerve. These data provide important information regarding the potential for quantitative morphometric changes associated with the use of nerve cuffs, and, importantly, suggests that these changes are associated with the degree of inflammation associated with the cuff.

An astrocyte derived extracellular matrix coating reduces astrogliosis surrounding chronically implanted microelectrode arrays in rat cortex.

Available evidence suggests that the magnitude of the foreign body response (FBR) to implants placed in cortical brain tissue is affected by the extent of vasculature damage following device insertion and the magnitude of the ensuing macrophage response. Since the extracellular matrix (ECM) serves as a natural hemostatic and immunomodulatory agent, we examined the ability of an FDA-approved neurosurgical hemostatic coating and an ECM coating derived from primary rat astrocytes to reduce the FBR surrounding a penetrating microelectrode array chronically implanted in rat cortex. Using quantitative methods, we examined various components of the FBR in vitro and after implantation. In vitro assays showed that both coatings accelerated coagulation in a similar fashion but only the astrocyte-derived material suppressed macrophage activation. In addition, the ECM coating derived from astrocytes, also decreased the astrogliotic response 8 weeks after implantation. Neither coating had a significant influence on the intensity or spatial distribution of FBR biomarkers 1 week after implantation or on degree of macrophage activation or neuronal survival at the later time point. The results show that microelectrode coatings with similar hemostatic properties but different immunomodulatory characteristics differentially affect the FBR to an anchored, single-shank, silicon microelectrode array. The results also support the concept that divergent biological pathways affect the various components of the FBR in the CNS and suggests that decreasing its impact will require a multifaceted approach.

The differential influence of colocalized and segregated dual protein signals on neurite outgrowth on surfaces.

We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (microCP) and microfluidic network (microFN) patterning to generate alternating stripes of pure single protein layers or (2) through microCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein-protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by microCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin-aggrecan interactions do not diminish laminin growth promoting properties.

The brain tissue response to implanted silicon microelectrode arrays is increased when the device is tethered to the skull.

The influence of tethering silicon microelectrode arrays on the cortical brain tissue reaction was compared with that of untethered implants placed in the same location by identical means using immunoflourescent methods and cell type specific markers over indwelling periods of 1-4 weeks. Compared with untethered, freely floating implants, tethered microelectrodes elicited significantly greater reactivity to antibodies against ED1 and GFAP over time. Regardless of implantation method or indwelling time, retrieved microelectrodes contained a layer of attached macrophages identified by positive immunoreactivity against ED1. In the tethered condition and in cases where the tissue surrounding untethered implants had the highest levels of ED1+ and GFAP+ immunoreactivity, the neuronal markers for neurofilament 160 and NeuN were reduced. Although the precise mechanisms are unclear, the present study indicates that simply tethering silicon microelectrode arrays to the skull increases the cortical brain tissue response in the recording zone immediately surrounding the microelectrode array, which signals the importance of identifying this important variable when evaluating the tissue response of different device designs, and suggests that untethered or wireless devices may elicit less of a foreign body response.

Astrocyte spreading and migration on aggrecan-laminin dot gradients.

The surface concentration gradient of two extracellular matrix (ECM) macromolecules was developed to study the migratory and morphological responses of astrocytes to molecular cues typically found in the central nervous system injury environment. The gradient, prepared using microcontact printing, was composed of randomly positioned micrometer-sized dots of aggrecan (AGG) printed on a substrate uniformly coated with laminin (LN). AGG dots were printed in an increasing number along the 1000 µm long and 50 µm wide gradient area which had on each end either a full surface coverage of AGG or LN. Each dot gradient was surrounded by a 100 µm-wide uniform field of AGG printed over laminin. Seeded astrocytes were found to predominantly attach to LN regions on the gradient. Cellular extensions of these cells were longer than the similar processes for cells seeded on uniform substrates of AGG or LN serving as controls. Astrocyte extensions were the largest and spanned a distance of 150 µm when the cells were attached to the mixed AGG+LN patches on the gradient. As evidenced by their increased area and perimeter, the cells extended processes in a stellate fashion upon initial attachment and maintained extensions when seeded in AGG+LN regions but not on uniform laminin controls. The cells migrated short distances, ∼20-35 µm, over 24 h and in doing so preferentially shifted from AGG areas to higher LN surface coverage regions. The results indicated that presenting mixed ECM cues caused astrocytes to sample larger areas of the substrate and made the cells to preferentially relocate to a more permissive ECM region.

Fabrication and characterization of permeable degradable poly(DL-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels.

Biodegradable permeable poly(DL-lactide-co-glycolide) (PLGA) hollow fiber membranes (HFMs) were fabricated using a wet phase inversion technique. By varying several parameters, such as the spinneret size, solvent and non-solvent pair, polymer concentration, flow rate, precipitation method, drop height, and small molecular pore-forming agents, PLGA HFMs with variable sizes, surface morphologies, porosities, and diffusive permeability were obtained. Under simulated physiological conditions in vitro, PLGA HFMs exhibited a degradation profile to accommodate nervous system regeneration and axonal outgrowth. While accelerated degradation resulted in substantial molecular weight loss starting at 2 weeks and loss of selective permeability at 3 weeks, PLGA HFMs maintained gross structural integrity in the first 4 weeks, followed by sharp weight loss at 6 weeks and complete disappearance at about 8 weeks. When compared to the raw PLGA material in a pellet form, which underwent heterogeneous degradation, the PLGA HFMs exhibited a homogeneous degradation where the surface and bulk degraded at approximately the same rate, and an overall lower degradation rate. Our results indicate that using a wet phase inversion technique, degradable HFMs with variable size, inner and outer surface morphologies, porosity, and permeability with potential applications for nerve tract guidance conduits can be fabricated.

Effect of filament diameter and extracellular matrix molecule precoating on neurite outgrowth and Schwann cell behavior on multifilament entubulation bridging device in vitro.

At present there is no clinically effective treatment for injuries or pathological processes that disrupt the continuity of axons in the mature central nervous system. However, a number of studies suggest that a tremendous potential exists for developing biomaterial based therapies. In particular, biomaterials in the form of bridging substrates have been shown to support at least some level of axonal regeneration across the lesion site, but display a limited capacity for directing axons toward their targets. To improve the directionality and outgrowth rate of the axonal regeneration process, filaments and tubes appear promising, but the technology is far from optimized. As a step toward optimization, the influence of filament diameter and various extracellular matrix coatings on nerve regeneration was evaluated in this article using a dorsal root ganglion (DRG) explant model. An increasing pattern of alignment and outgrowth of neurites in the direction parallel the long axis of the packed filament bundles with decreasing filament diameters ranging from supracellular and beyond (500 to 100 mum), cellular (30 mum), down to subcellular size (5 mum) was observed. Such effects became most prominent on filament bundles with individual filament diameters in the range of cellular size and below (5 and 30 mum) where highly directional and robust neuronal outgrowth was achieved. In addition, laminin-coated filaments that approached the size of spinal axons support significantly longer regenerative outgrowth than similarly treated filaments of larger diameter, and exceed outgrowth distance on similarly sized filaments treated with fibronectin. These data suggested the feasibility of using a multifilament entubulation bridging device for supporting directional axonal regeneration.

Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs.

Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25 Hz, 8 h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFbeta-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production.

Acute microelectrode array implantation into human neocortex: preliminary technique and histological considerations.

OBJECT: Researchers at The Center for Neural Interfaces at the University of Utah have designed and produced a silicon-based high-density microelectrode array that has been used successfully in mammalian models. The authors investigate the ability to transfer array insertion techniques to humans and examine the acute response of human cortical tissue to array implantation. METHODS: Six patients who were scheduled to undergo temporal lobectomy surgery were enrolled in an Institutional Review Board-approved protocol. Before the patients underwent lateral temporal cortical resection, one or two high-density microelectrode arrays were implanted in each individual by using a pneumatic insertion device. Cortical tissue was then excised and preserved in formalin. The specimens were sectioned and stained for histological examination. Pneumatic insertion of a microelectrode array into human cortex in the operating room was feasible. There were no clinical complications associated with implantation and no evidence of significant insertion-related hemorrhage. Tissue responses ranged from mild cortical deformity to small focal hemorrhages several millimeters below the electrode tines. Based on initial results, the insertion device was modified. A footplate that mechanically isolates a small area of cortex and a calibrated micromanipulator were added to improve the reproducibility of insertion. CONCLUSIONS: A high-density microelectrode array designed to function as a direct cortical interface device can be implanted into human cortical tissue without acute clinical complications. Further modifications to the insertion device and array design are ongoing and future work will assess the functional significance of the tissue reactions observed.

Directional neurite outgrowth is enhanced by engineered meningeal cell-coated substrates.

After injury to the CNS, the anatomical organization of the tissue is disrupted, posing a barrier to the regeneration of axons. Meningeal cells, a central participant in the CNS tissue response to injury, migrate into the core of the wound site in an unorganized fashion and deposit a disorganized extracellular matrix (ECM) that produces a nonpermissive environment. Previous work in our laboratory has shown that the presentation of nanometer-scale topographic cues to these cells influences their morphological, cytoskeletal, and secreted ECM alignment. In the present study, we provided similar environmental cues to meningeal cells and examined the ability of the composite construct to influence dorsal root ganglion regeneration in vitro. When grown on control surfaces of meningeal cells lacking underlying topographic cues, there was no bias in neurite outgrowth. In contrast, when grown on monolayers of meningeal cells with underlying nanometer-scale topography, neurite outgrowth length was greater and was directed parallel to the underlying surface topography even though there exists an intervening meningeal cell layer. The observed outgrowth was significantly longer than on laminin-coated surfaces, which are considered to be the optimal substrata for promoting outgrowth of dorsal root ganglion neurons in culture. These results suggest that the nanometer-level surface finish of an implanted biomaterial may be used to organize the encapsulation tissue that accompanies the implantation of materials into the CNS. It furthermore suggests a simple approach for improving bridging materials for repair of nerve tracts or for affecting cellular organization at a device-tissue interface.

Response of brain tissue to chronically implanted neural electrodes.

Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants, making the biocompatibility of implanted electrodes a primary concern in device design. This review presents the biological components and time course of the acute and chronic tissue reaction in brain tissue, analyses the brain tissue response of current electrode systems, and comments on the various material science and bioactive strategies undertaken by electrode designers to enhance electrode performance.

A cell encapsulation device for studying soluble factor release from cells transplanted in the rat brain.

The transplantation of a variety of naturally occurring and genetically modified cell types has been shown to be an effective experimental method to achieve sustained delivery of therapeutic molecules to specific target areas in the brain. To acquire a better understanding of dosing, implant mechanism of action, and how certain cell types affect remodeling of central nervous system (CNS) tissue, a refillable cell encapsulation device was developed for introducing cells into the brain while keeping them physically isolated from contact with brain tissue with a semipermeable membrane. The stereotactically placed device consists of a hollow fiber membrane (HFM), a polyurethane grommet with watertight cap that snaps into a precisely drilled hole in the rat skull, and a removable cell-containing insert. The cell-containing insert can be introduced or removed in a time-dependent manner to study the influence of soluble factors released from transplanted cells. The study describes the device design and validates its utility using a well-established cell transplantation model of Parkinson's disease.

Impedance characterization of microarray recording electrodes in vitro.

The mechanisms underlying performance degradation of chronically implanted silicon microelectrode arrays in the central nervous system (CNS) remain unclear. Humoral and cellular components of the brain foreign body response were evaluated to determine whether their presence on the electrode surface results in increased electrical impedance. Iridium oxide microelectrode recording arrays were electrically characterized in saline, culture media with 10% fetal bovine serum, and coated with various CNS cell types isolated from rat brain. Electrochemical impedance spectroscopy and cyclic voltammetry were performed using a three-electrode system. Potential cycling caused an immediate decrease in electrical impedance, which increased with time toward precycling values, with the effect of cycling remaining significant for several days. The addition of serum caused a significant increase in impedance of up to 28% relative to the saline control. Microelectrodes coated with various cell types known to participate in the foreign body response caused a 20%-80% increase in impedance immediately after contact that remained constant or gradually increased for several weeks. Our findings suggest that the attachment of molecular and cellular species following microelectrode implantation into brain tissue likely contribute to increases in impedance, but do not appear sufficient to hinder recording performance.

Differences Exist in the Left and Right Sciatic Nerves of Naïve Rats and Cats.

The sciatic nerve of rats and cats is commonly used in experimental models of peripheral nerve injury and repair, as well as experiments involving peripheral nerve electrode implantation. In such experiments, morphometric parameters from the implanted nerve are commonly evaluated and compared to control values obtained from the contralateral nerves. However, this may not be an appropriate approach as differences may naturally exist in the structure of the two nerves owing to developmental or behavioral asymmetry. Additionally, in the cat, baseline values for standard morphometric parameters are not well established. In this study, we characterized fascicle area, fiber count, fiber density, fiber packing, mean g-ratio, and fiber diameter distributions in the rat and cat, as well as investigated the potential for naturally occurring sided differences in these parameters in both species. We also investigated whether animal age or location along the nerve influenced these parameters. We found that sided or left/right leg differences exist in some parameters in both the rat and the cat, calling into question the validity of using the contralateral nerve as a control. We also found that animal age and location along the nerve can make significant differences in the parameters tested, establishing the importance of using control nerves from age- and behaviorally matched animals whose morphometric parameters are collected and compared from the same location.