Mechanically-adaptive, resveratrol-eluting neural probes improve single-unit recording performance

Introduction:: Neural probes, or intracortical microelectrodes, are used as a part of brain-computer interface systems to restore motor and sensory function in individuals with spinal cord injury, limb loss, and neurodegenerative disorders^1,2. Unfortunately, they fail shortly after implantation, characterized by reduced neural recording performance, resulting in decreased therapeutic efficacy^3-5. Failure is attributed to an increased neuroinflammatory response, distinguished by a state of oxidative stress and mechanical mismatch at the device-tissue interface^6,7. Oxidative stress can perpetuate the foreign body response, leading to an elevated amount of activated microglia and reactive oxygen species around the implant site, neuronal death, and corrosion of the electrode contacts^8,9. Mechanical mismatch at the device-tissue interface can lead to increased differential strain, resulting in inflammation and glial scar encapsulation¹⁰. Local delivery of antioxidant and anti-inflammatory therapeutics, such as resveratrol, and mechanically-compliant materials have been shown to reduce neuroinflammatory markers and improve neuron density around the implant site^11-13. However, previous microfabrication limitations have prevented the addition of functional recording electrodes on the substrate, providing little insight about their effect on neural recording performance. In this work, we utilized a unique microfabrication process to integrate functional recording electrodes on the substrate. Additionally, we evaluated the mechanically-adaptive, resveratrol-eluting (MARE) neural probes in an in vivo model to compare neural recording performance and the neuroinflammatory response to traditional silicon probes. Our hypothesis is that the MARE neural probes will show improved recording performance and reduced neuroinflammation when compared to silicon devices.


Materials and Methods:: The mechanically-adaptive substrate is a polymer-based nanocomposite, polyvinyl acetate with tunicate cellulose nanocrystals, loaded with resveratrol. The material is created by a dispersion, casting, and evaporation method, resulting in a resveratrol-loaded thin film substrate. Microfabrication techniques were utilized to integrate functional recording electrodes on the substrate as follows¹⁴. First, a Parylene C barrier layer was vapor-deposited on both sides of the substrate, followed by sputter-deposition of a Ti adhesion layer and Au conductive layer. The metal was patterned with photolithography and excess was wet-etched. A second Parylene C insulation layer was vapor-deposited and patterned with oxygen plasma. Finally, individual probes were laser-micromachined. Probes were packaged for interfacing with electronics for neural recording. Resveratrol release profiles were established by incubating the resveratrol-loaded material in 1X PBS in a water bath incubator at 37˚C. At a designated timepoint, a sample of the incubation solution was measured using UV-vis spectrophotometry to determine the amount of resveratrol released. Devices were then tested with electrochemical impedance spectroscopy (EIS) to ensure that the electrode impedance at 1 kHz was in a suitable range for extracellular neural recording. Probes were then subject to in vivo testing in Sprague Dawley rats. Rats were implanted with either a MARE probe (experimental) or silicon probe (control) in the motor cortex following existing lab surgical procedures. Neural recording and in vivo EIS sessions were done biweekly following implantation. At 4 weeks, rats underwent a cardiac perfusion and the brain was extracted and frozen for further analysis of genetic and proteomic markers.


Results, Conclusions, and Discussions:: MARE probes with functional recording electrodes can be successfully fabricated and packaged for in vivo neural recording applications, as shown in Figure 1. Resveratrol release experiments showed the release profiles of films loaded at two different resveratrol concentrations, 0.01% and 1% w/w, to determine the release profile and timeline over a 67-day time period, shown in Figure 2. There is a fast initial release over the first 60 hours, with slower release continuing thereafter over the entire experiment duration, showing that the material is capable of controlled release, making it optimal for in vivo use. Additionally, devices evaluated with EIS prior to implantation showed 1 kHz impedances in the range for extracellular neural recording, between 100-1000 kΩ. Figure 3 shows a representative benchtop EIS spectra from a channel of a MARE probe, with the |Z| at 1 kHz of 166 kΩ, and a dynamic phase response. Imaging and EIS testing assured that the MARE probes were capable of neural recording and fit for in vivo testing. MARE probes in Sprague Dawley rats were able to record single unit action potentials, as shown in the pile plot in Figure 4. The pile plot shows all waveforms associated with a single unit in gray lines, with the average waveform of the single unit shown with a black line. The peak-to-peak amplitude is 102 μV and the signal-to-noise ratio is 8.9, favorable characteristics for single unit recording. When comparing MARE and silicon recording performance using the percentage of active channels – the percentage of channels recording at least 1 single unit – MARE probes showed improvement over silicon probes. 73% of MARE probe channels were active at 4 weeks, compared to just 52% of silicon probe channels, shown in Figure 5. This work has shown that MARE probes can be fabricated, tested, and implemented in vivo with improved recording performance and longevity compared to the current industry standard, silicon probes. As the field pursues better therapeutic efficacy for patients with neuromusculoskeletal disorders, utilizing devices like MARE probes that mitigate the neuroinflammatory response to improve recording performance over time is critical for clinical implementation.


Acknowledgements (Optional): : This work was supported by Merit Review Award #12647351 (Hess-Dunning/Capadona) from the United States Department of Veterans Affairs Rehabilitation Research and Development Service and the FY2021 National Defense Science and Engineering Graduate Fellowship Program.


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