Ultrasoft microwire neural electrodes improve chronic tissue integration

Abstract

Chronically implanted neural multi-electrode arrays (MEA) are an essential technology for recording electrical signals from neurons and/or modulating neural activity through stimulation. However, current MEAs, regardless of the type, elicit an inflammatory response that ultimately leads to device failure. Traditionally, rigid materials like tungsten and silicon have been employed to interface with the relatively soft neural tissue. The large stiffness mismatch is thought to exacerbate the inflammatory response. In order to minimize the disparity between the device and the brain, we fabricated novel ultrasoft electrodes consisting of elastomers and conducting polymers with mechanical properties much more similar to those of brain tissue than previous neural implants. In this study, these ultrasoft microelectrodes were inserted and released using a stainless steel shuttle with polyethyleneglycol (PEG) glue. The implanted microwires showed functionality in acute neural stimulation. When implanted for 1 or 8weeks, the novel soft implants demonstrated significantly reduced inflammatory tissue response at week 8 compared to tungsten wires of similar dimension and surface chemistry. Furthermore, a higher degree of cell body distortion was found next to the tungsten implants compared to the polymer implants. Our results support the use of these novel ultrasoft electrodes for long term neural implants.

Statement of significance: One critical challenge to the translation of neural recording/stimulation electrode technology to clinically viable devices for brain computer interface (BCI) or deep brain stimulation (DBS) applications is the chronic degradation of device performance due to the inflammatory tissue reaction. While many hypothesize that soft and flexible devices elicit reduced inflammatory tissue responses, there has yet to be a rigorous comparison between soft and stiff implants. We have developed an ultra-soft microelectrode with Young's modulus lower than 1MPa, closely mimicking the brain tissue modulus. Here, we present a rigorous histological comparison of this novel ultrasoft electrode and conventional stiff electrode with the same size, shape and surface chemistry, implanted in rat brains for 1-week and 8-weeks. Significant improvement was observed for ultrasoft electrodes, including inflammatory tissue reaction, electrode-tissue integration as well as mechanical disturbance to nearby neurons. A full spectrum of new techniques were developed in this study, from insertion shuttle to in situ sectioning of the microelectrode to automated cell shape analysis, all of which should contribute new methods to the field. Finally, we showed the electrical functionality of the ultrasoft electrode, demonstrating the potential of flexible neural implant devices for future research and clinical use.

Keywords: Composite bio-electrodes; Conducting elastomer; Deep brain stimulation; Neural electrode.

Fig. 1

Illustration of fabricated parts, implant assembly, insertion and wire electrode release. From top: 1) Original needle; 2) Fabricated half-needle shuttle; 3) Mount soft/ stiff wire; 4) PEG facilitated assembly; 5) Wire detachment in vivo. Inset on top right corner: SEM image of fabricated soft wire tip. Scale bar = 100 µm.

Fig. 2

NF200, Iba-1 and GFAP expression around soft and stiff implants. NF200, Iba-1 and GFAP stain for axons, microglia and astrocytes, respectively. M-P are the overlay images including Hoechst. Groups of histology from left-most are soft wire at week-1, stiff wire at week-1, soft wire at week-8 and stiff wire at week-8. These groups are consistent through all immunohistochemistry figures. Scale bar = 100 µm. Q and R are the normalized intensity values for NF-200 expression at week-1 (animal n = 7, multiple samples from one animal, outliers removed according to 2*standard deviation, N = 42 histological samples quantified for soft wire, for stiff N = 42. All subsequent are denoted similarly.) and week-8(soft N = 34, stiff N = 38), respectively; S and T are normalized intensity values for Iba-1 expression at week-1(soft N = 45, stiff N = 46) and week-8(soft N = 34, stiff N = 35), respectively; U and V are normalized intensity values for GFAP expression at week-1(soft N = 45, stiff N = 43) and week-8(soft N = 33, stiff N = 37), respectively. Intensities are calculated 0 µm from the electrode-tissue interface until 300 µm away, with 5 µm bin size.

Fig. 3

Apoptotic cell death around soft and stiff implant. Cleaved Caspase 3 (Casp3) indicates apoptotic cell death and NeuN indicates neurons. Very little co-localization of Casp3 and NeuN was observed in any group. I-L are the overlay of these images with Hoechst. Scale bar = 100 µm. M and N are cell density counts per mm² at week-1(soft N = 14, stiff N = 13) and week-8(soft N = 10, stiff N = 11), respectively; cell counts were calculated by automated analysis of NeuN staining and the result was binned 30 µm from the electrode-tissue interface until 210 µm away, with 30 µm bin size. Bins start 30 µm from the interface because cell count within 30 µm of the interface were too low, yielding inaccurate results.

Fig. 4

Neuronal cell deformation around soft and stiff implants. A. Example image of NeuN staining for week-8 around a stiff implant. Neurons on the left side of the image show significant distortion possibly caused by the micro-motion related strain from the implant. Scale bar = 100 µm. B. Illustration of CEA and CSSI definitions exemplified by a post-processed cell image. Automatical segmentation yielded the red dots as the pixel boundary of the cell. The dashed green line is the long axis direction, the solid green line is the length of the long axis b, the solid yellow line is the length of the short axis a, and the solid blue line is the connection between the center of the cell and the center of the implanted wire. The cyan arc depicts the definition of CEA as the angle between the dashed green line and the solid blue line. Scale bar = 5 µm. C and E are CEA comparisons between soft and stiff wires at week-1 and week-8, respectively. D and F are CSSI comparisons between soft and stiff wires at week-1 and week-8, respectively. The dashed gray line in each image represents the mean of the CEA or CSSI analysis result from control images in non implanted cortical tissue sections. All results are binned 30 µm from the electrode-tissue interface until 210 µm away, with 30 µm bin size.

Fig. 5

Chronic BBB leakage and potential neural regeneration around implants. Vimentin (green) clearly aggregates around both implant types although the stiff wires cause a broad range increase of vimentin expression far from the implant as well. L1 (red) is associated with tissue response as well as possible developing oligodendrocytes for axon repair. Very strong L1 activity is observed around stiff implants at both time points. IgG (gray) indicates BBB leakage. IgG intensity around the stiff implants was very similar to that around soft wires at week 1 but became much stronger at week 8, indicating a significant chronic leakage of the BBB caused by the stiff implant. Scale bar = 100 µm. Q and R are normalized intensity values for Vimentin at week-1(soft N = 9, stiff N = 8) and week-8(soft N = 8, stiff N = 10), respectively; S and T are normalized intensity values for L1(week-1 soft N = 10, stiff N = 8, week-8 soft N = 9, stiff N = 10); U and V are normalized intensity values for IgG(week-1 soft N = 10, stiff N = 9, week-8 soft N = 10, stiff N = 11). Intensities are calculated 0 µm from the electrode-tissue interface until 300 µm away, with 5 µm bin size. Q and S demonstrate a broad range increase of Vimentin and L1 around stiff implants at week-1. R, T and V all indicate that a significant portion of the chronic tissue response was caused by the mechanical properties of the stiff implant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Cell adhesion and surface morphology of explanted soft and stiff implants. Hochest (blue), beta-III tubulin (green) and Iba-1 (red) exhibit uniform and confluent coverage on soft wires at week-1 and week-8 with dominant beta-III tubulin expression. In comparison, the cells on stiff wires are scattered with Iba-1 domanance especially at week-1, indicating the stiff wires have poor integration with tissue and that microglia cells are preferentially attracted to this surface. Each image is the reconstruction of 11 z-stack confocal microscope images. Scale bar = 100 µm. Q-T illustrate the wire morphology under SEM imaging. The insulation layer on the stiff wire is visible at both week-1 and week-8. At week-8, the soft wire not only attracted a layer of cells, but also exhibited a bulky tissue shell consisting mostly of neural cells according to beta-III tubulin intensity in G and overlay image O. SEM scale bar = 5 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Impedance and DBS performance of the elastomer electrode. A. Impedance spectrum of the elastomer electrode for neural stimulation. A thin layer of gold is sputtered on the side of the conductive elastomer core to provide better conductivity. Very low impedance is recorded on the polymer electrode to prove the neural stimulation capability. B. STN DBS with the soft wire evoked a strong LFP in the ipsilateral motor cortex recorded by a ground screw EEG. Stars denote the electrical stimulation artifacts.