Biocompatibility assessment of insulating silicone polymer coatings using an in vitro glial scar assay.

Vapor-deposited silicone coatings are attractive candidates for providing insulation in neuroprosthetic devices owing to their excellent resistivity, adhesion, chemical inertness and flexibility. A biocompatibility assessment of these coatings is an essential part of the materials design process, but current techniques are limited to rudimentary cell viability assays or animal muscle implantation tests. This article describes how a recently developed in vitro model of glial scar formation can be utilized to assess the biocompatibility of vapor-deposited silicone coatings on micron-scale wires. A multi-cellular monolayer comprising mixed glial cells was obtained by culturing primary rat midbrain cells on poly(D-lysine)-coated well plates. Stainless steel microwires were coated with two novel insulating thin film silicone polymers, namely poly(trivinyltrimethylcyclotrisiloxane) (polyV(3)D(3)) and poly(trivinyltrimethylcyclotrisiloxane-hexavinyldisiloxane) (polyV(3)D(3)-HVDS) by initiated chemical vapor deposition (iCVD). The monolayer of midbrain cells was disrupted by placing segments of coated microwires into the culture followed by immunocytochemical analysis after 7 d of implantation. Microglial proximity to the microwires was observed to correlate with the amount of fibronectin adsorbed on the coating surface; polyV(3)D(3)-HVDS adsorbed the least amount of fibronectin compared to both stainless steel and polyV(3)D(3). Consequently, the relative number of microglia within 100 µm of the microwires was least on polyV(3)D(3)-HVDS coatings compared to steel and polyV(3)D(3). In addition, the astrocyte reactivity on polyV(3)D(3)-HVDS coatings was lower compared to stainless steel and polyV(3)D(3). The polyV(3)D(3)-HVDS coating was therefore deemed to be most biocompatible, least reactive and most preferable insulating coating for neural prosthetic devices.

Soluble factor effects on glial cell reactivity at the surface of gel-coated microwires.

A basal lamina gel preparation was incorporated into a modified neuroinflammation cell culture model to test the system as a characterization tool for surface-modified microwires. The extent of gliosis at the surface of gel-coated microwires was quantified in response to titrating the cell culture with a number of soluble factors reported to be involved in reactive gliosis. Positive control conditions (1% FBS, 10 ng/ml bFGF, Neural Basal medium with B27 supplement) induced a layer of GFAP-expressing astrocytes to accumulate on all gel-coated microwires. Serum, inflammatory cytokines IL-1alpha and IL-1beta and the neural progenitor cell (NPC) proliferating growth factors bFGF and PDGF all increased the number of reactive cells on the gel, in agreement with their reported roles in cell activation, migration and proliferation at the site of injury. Technically, this study shows that a fetal neuron-glia culture system is well suited for characterizing the impact of electrode coatings designed to mitigate implant-associated gliosis, and for screening the effect of multiple soluble factors that promote and/or inhibit implant-associated gliosis. Scientifically, this study points to essential roles of serum and inflammatory factors to induce NPC activation and migration to the site of injury, where growth factors like bFGF and PDGF induce proliferation of cells that will eventually form the glial scar.

Reactive microgliosis: extracellular micro-calpain and microglia-mediated dopaminergic neurotoxicity.

Microglia, the innate immune cells in the brain, can become chronically activated in response to dopaminergic neuron death, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis), which is implicated in the progressive nature of Parkinson's disease. Here, we use an in vitro approach to separate neuron injury factors from the cellular actors of reactive microgliosis and discover molecular signals responsible for chronic and toxic microglial activation. Upon injury with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium, N27 cells (dopaminergic neuron cell line) released soluble neuron injury factors that activated microglia and were selectively toxic to dopaminergic neurons in mixed mesencephalic neuron-glia cultures through nicotinamide adenine dinucleotide phosphate oxidase. mu-Calpain was identified as a key signal released from damaged neurons, causing selective dopaminergic neuron death through activation of microglial nicotinamide adenine dinucleotide phosphate oxidase and superoxide production. These findings suggest that dopaminergic neurons may be inherently susceptible to the pro-inflammatory effects of neuron damage, i.e. reactive microgliosis, providing much needed insight into the chronic nature of Parkinson's disease.

Control protocol for robust in vitro glial scar formation around microwires: essential roles of bFGF and serum in gliosis.

Previously, we reported an in vitro cell culture model that recreates many of the hallmarks of glial scarring around electrodes used for recording in the brain; however, the model lacked the reproducibility necessary to establish a useful characterization tool. This methods paper describes a protocol, modeled on protocols typically used to culture neural stem/precursor cells, that generates a predictable positive control of an intense scarring reaction. Six independent cell culture variables (growth media, seeding density, bFGF addition day, serum concentration in treatment media, treatment day, and duration of culture) were varied systematically and the resulting scars were quantified. The following conditions were found to give the highest level of scarring: Neurobasal medium supplemented with B27, 10% fetal bovine serum at treatment, 10 ng/ml b-FGF addition at seeding and at treatment, treatment at least 6 days after seeding and scar growth of at least 5 days. Seeding density did not affect scarring as long as at least 500,000 cells were seeded per well, but appropriate media, bFGF, and serum were essential for significant scar formation-insights that help validate the in vitro-based approach to understanding glial scarring. With the control protocol developed in this study producing a strong, reproducible glial scarring positive control with every dissection, this culture model is suitable for the in vitro study of the mechanisms behind glial scarring and neuroelectrode failure.

In vitro model of glial scarring around neuroelectrodes chronically implanted in the CNS.

A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-D-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 microm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Vimentin expressing cells and processes were present in the scrape at early times and within the astrocyte processes forming the glial scar. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording. The time course and relative positions of the glia in response to the different injury paradigms correlated well with stereotypical in vivo responses and warrant further work in the development of a functional in vitro test bed.

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.