Biocompatibility of a polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+) for neural implants.

The flexibility of implantable neural probes has increased during the last 10 years, starting with stiff materials such as silicone to more flexible materials like polyimide. We have developed a novel polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+, consisting of a thiol, two allyls, an epoxy resin and two initiators), which is up to 100 times more flexible than polyimide. Since a flexible neural probe should be more biocompatible than a stiff probe, an OSTE+ probe should be more biocompatible than one composed of a more rigid material. We have investigated the toxicity of OSTE+ as well as of OSTE+ that had been incubated in water for a week (OSTE+H2O) using MTT assays with mouse L929 fibroblasts. We found that OSTE+ showed cytotoxicity, but OSTE+H2O did not. Extracts were analyzed using LC-MS and GC-MS in order to identify leaked chemicals

Development of a polymer based neural probe - How to record intracortical neural activity while minimizing the tissue response.

Intracortical neural probes have in the last couple of years been developed from stiff single probes to stiff multi-electrode neural probes to flexible multi-electrode neural probes. One reason for the change in design is that more than one recording/stimulation electrode, as in the case with a single wire, is needed for more advanced studies. The stiff multi-electrode probes were fabricated using silicon as the base and the stiffness of silicon made the probes unable to follow the micro motions of the brain. This led to the development of polymer based multi-electrode probes that are better to follow the micro motions of the brain. The polymer based neural probes, when comparing to a stiff probe, have less tissue response, reduced encapsulation of the probe by glial cells, increased functional neurons around the probe and a smaller void around the probe in the tissue. It has also been shown that the encapsulation around the neural probe is the largest around the base of the probe, while at the edges the encapsulation is smaller. When designing a neural probe used for neural recordings, it is of interest to keep the electrical impedance of the interface between the electrode and the tissue as small as possible, since it will generate a signal with a better signal to noise ratio than if the electrical impedance is large. The impedance can be decreased by increasing the size of the electrode, but if the size of the electrode gets too large it might act as a direct connection between two neurons, hence the size should be as small as possible. The solution to this is to modify the electrode surface to become porous/rough, which will keep the geometrical size of the electrode small and at the same time keep the active area of the electrode large. With all this in mind, my work has mainly been about designing a flexible polymer based intra cortical neural probe for chronic recordings. The probe, designed by me and my colleges, reassembles a flat and flexible Christmas tree and on the tip of each branch there is an electrode. There are 9-13 platinum black modified recording electrodes on the probe. The platinum black modification decreases the electrical impedance of each electrode by roughly one order of magnitude. In one study the probe recorded neural activity from mossy fibers and climbing fibers in the cerebellar molecular layer in rats. In one study it was used to analyze how hyperalgesia can be seen in the somatosensory cortex of free moving rats. Not only has the overall design of the probe been developed over time, but also the material out of which the probe was constructed. The polymer has been changed first from SU-8 to polyimide, and then to a newly developed polymer called OSTE+. I further developed OSTE+ from it's original use in micro fluidic devices. Polyimide has the benefit over SU-8 that it is not as brittle and OSTE+ has the benefit over polyimide and SU-8 that it can be up to 500 times more flexible. Since OSTE+ is a newly developed polymer two biocompatibility studies were done; the first one used in vitro MTT assays together with mass spectroscopy for analyzing the biocompatibility and in the second one in vitro immunohistochemistry was used for the biocompatibility studies. It was shown that OSTE+ is rendered nontoxic to cells if it is incubated in water for one-week prior to use and that the tissue response of OSTE+ compared to polyimide is similar

A polymer based electrode array for recordings in the cerebellum

A polymer foil based array with 9 gold electrodes for chronic electrophysiological recordings in the CNS has been developed. A polymer, SU-8, is pattered using photolithography techniques used every day in micro fabrication. This is beneficial if the electrode would be manufactured on a large scale basis. The technique makes it easy to adapt the array to best fit the structure of interest at the time, in this study the rat cerebellar cortex. The use of SU-8 as the base of the array makes the array very flexible, hence lets it stay close to the same cells following the movement of the brain. The electrodes can then be modified with platinum black to lower the impedance of the electrode up to one order of magnitude, making us able to create smaller electrodes but keeping the low impedance necessary to get a get the signal to noise ratio required. Platinum black modified arrays were also implanted chronically and showed excellent signal recording capabilities in rat cerebellum

μ-Foil Polymer Electrode Array for Intracortical Neural Recordings

We have developed a multichannel electrode array—termed μ-foil—that comprises ultrathin and flexible electrodes protruding from a thin foil at fixed distances. In addition to allowing some of the active sites to reach less compromised tissue, the barb-like protrusions that also serves the purpose of anchoring the electrode array into the tissue. This paper is an early evaluation of technical aspects and performance of this electrode array in acute in vitro/in vivo experiments. The interface impedance was reduced by up to two decades by electroplating the active sites with platinum black. The platinum black also allowed for a reduced phase lag for higher frequency components. The distance between the protrusions of the electrode array was tailored to match the architecture of the rat cerebral cortex. In vivo acute measurements confirmed a high signal-to-noise ratio for the neural recordings, and no significant crosstalk between recording channels

Influence of probe flexibility and gelatin embedding on neuronal density and glial responses to brain implants.

To develop long-term high quality communication between brain and computer, a key issue is how to reduce the adverse foreign body responses. Here, the impact of probe flexibility and gelatine embedding on long-term (6w) tissue responses, was analyzed. Probes of same polymer material, size and shape, flexible mainly in one direction, were implanted in rat cerebral cortex (nimplants = 3 x 8) in two orientations with respect to the major movement direction of the brain relative to the skull: parallel to (flex mode) or transverse to (rigid mode). Flex mode implants were either embedded in gelatin or non-embedded. Neurons, activated microglia and astrocytes were visualized using immunohistochemistry. The astrocytic reactivity, but not microglial response, was significantly lower to probes implanted in flex mode as compared to rigid mode. The microglial response, but not astrocytic reactivity, was significantly smaller to gelatin embedded probes (flex mode) than non-embedded. Interestingly, the neuronal density was preserved in the inner zone surrounding gelatin embedded probes. This contrasts to the common reports of reduced neuronal density close to implanted probes. In conclusion, sheer stress appears to be an important factor for astrocytic reactivity to implanted probes. Moreover, gelatin embedding can improve the neuronal density and reduce the microglial response close to the probe

A polymer neural probe with tunable flexibility

A novel polymeric material, off-stoichiometry thiol-ene-epoxy (OSTE+), has been evaluated for the fabrication of neural implants. OSTE+ is easily photo-structurable and exhibits mechanical properties suitable for stable implantation of the probe into brain tissue, while being sufficiently soft at physiological temperatures to reduce living tissue damage. The facile processing of OSTE+ allows use in applications where SU-8 or polyimide currently are the materials of choice. Uniquely, OSTE+ has a Young's modulus of 1.9 GPa at 10 degrees C decreasing almost two orders of magnitude to 30 MPa at 40 degrees C, which can be compared to the Young's modulus of 2.1 GPa for SU-8. We show a probe, with nine gold electrode sites, implanted into 0.5% agar at 40 degrees C using active cooling during the implantation

Histological evaluation of flexible neural implants; Flexibility limit for reducing the tissue response?

Objective. Flexible neural probes are hypothesized to reduce the chronic foreign body response (FBR) mainly by reducing the strain-stress caused by an interplay between the tethered probe and the brain's micromotion. However, a large discrepancy of Young's modulus still exists (3-6 orders of magnitude) between the flexible probes and the brain tissue. This raises the question of whether we need to bridge this gap; would increasing the probe flexibility proportionally reduce the FBR? Approach. Using novel off-stoichiometry thiol-enes-epoxy (OSTE+) polymer probes developed in our previous work, we quantitatively evaluated the FBR to four types of probes with different softness: silicon (∼150 GPa), polyimide (1.5 GPa), OSTE+Hard (300 MPa), and OSTE+Soft (6 MPa). Main results. We observed a significant reduction in the fluorescence intensity of biomarkers for activated microglia/macrophages and blood-brain barrier (BBB) leakiness around the three soft polymer probes compared to the silicon probe, both at 4 weeks and 8 weeks post-implantation. However, we did not observe any consistent differences in the biomarkers among the polymer probes. Significance. The results suggest that the mechanical compliance of neural probes can mediate the degree of FBR, but its impact diminishes after a hypothetical threshold level. This infers that resolving the mechanical mismatch alone has a limited effect on improving the lifetime of neural implants

Visualization of the probe used for implantation, implantation modes and regions of interest for image analysis.

<p>A) Schematic overview of the probe. Images are not to scale. B) Shanks were inserted 1 mm caudal and 2.3 mm lateral to Bregma (B, Bregma), either sagittal (rigid mode, 1) or coronal (flex mode, 2). (L, Lambda). C) Two regions of interests (ROIs) were analyzed in NIS Elements 3.1 software (Nikon, Japan), measuring 0–50 μm (inner ROI, marked with a red line) and 50–200 μm (outer ROI, marked with a green line) from the estimated border of the hole left by the implant.</p

Effects on neuronal density and morphology.

<p>A) Representative images of rigid, flex and flex embedded mode implantations after 6 weeks, showing mode dependent alterations neuronal densities, as shown in green (NeuN), total cell nuclei (DAPI) in blue and merged (GFAP in red depicting the implantation scar). Scale bar: 50 μm. B and C) Quantification of the NeuN and DAPI densities, respectively, that surrounds the different implantation sites at the inner ROI (0–50 μm). Note that in B) the NeuN densities are compared to paired naïve areas (controls). The box corresponds to the 25th and 75th percentiles, the median value is indicated by the horizontal line within each box, and the whiskers show the minimum and maximum values. The horizontal lines indicate statistical differences.</p

Implantation mode and embedding dependent glial reactions.

<p>A) Representative images of rigid, flex and flex embedded mode implantations after 6 weeks, showing mode dependent alterations in astrocytosis, as shown in red (GFAP), microglia responses, as shown in green (ED1) and merged (DAPI in blue (total cell nuclei)). Scale bar: 50 μm. Cephalocaudal direction corresponds to top-to-bottom in image. B and C) Quantification of the GFAP and ED1 densities, respectively, that surrounds the different implantation sites in the inner ROI (0–50 μm). The box corresponds to the 25th and 75th percentiles, the median value is indicated by the horizontal line within each box, and the whiskers show the minimum and maximum values. The horizontal lines indicate statistical differences.</p