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[Aims/hypothesis] A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents., [Methods] Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O2 + 90% N2). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT., [Results] KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [KITT] HFHSu sham, 2.56 ± 0.41% glucose/min; KITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks., [Conclusions/interpretation] KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans., This study was supported financially by Galvani Bioelectronics (formerly the Bioelectronics R&D unit at GlaxoSmithKline). JFS and BFM are supported by PhD Grants from the Portuguese Foundation for Science and Technology (reference PD/BD/105890/2014 and PD/BD/128336/2017, respectively). Data in the present manuscript has been filed with the International Bureau WO/2016/72875. International application no. PCT/PT2015/000047.

The nascent field of bioelectronic medicine seeks to decode and modulate peripheral nervous system signals to obtain therapeutic control of targeted end organs and effectors. Current approaches rely heavily on electrode-based devices, but size scalability, material and microfabrication challenges, limited surgical accessibility, and the biomechanically dynamic implantation environment are significant impediments to developing and deploying advanced peripheral interfacing technologies. Here, we present a microscale implantable device – the nanoclip – for chronic interfacing with fine peripheral nerves in small animal models that begins to meet these constraints. We demonstrate the capability to make stable, high-resolution recordings of behaviorally-linked nerve activity over multi-week timescales. In addition, we show that multi-channel, current-steering-based stimulation can achieve a high degree of functionally-relevant modulatory specificity within the small scale of the device. These results highlight the potential of new microscale design and fabrication techniques for the realization of viable implantable devices for long-term peripheral interfacing.

Objective. Most preparations for making neural recordings degrade over time and eventually fail due to insertion trauma and reactive tissue response. The magnitudes of these responses are thought to be related to the electrode size (specifically, the cross-sectional area), the relative stiffness of the electrode, and the degree of tissue tolerance for the material. Flexible carbon fiber ultra-microelectrodes have a much smaller cross-section than traditional electrodes and low tissue reactivity, and thus may enable improved longevity of neural recordings in the central and peripheral nervous systems. Only two carbon fiber array designs have been described previously, each with limited channel densities due to limitations of the fabrication processes or interconnect strategies. Here, we describe a method for assembling carbon fiber electrodes on a flexible polyimide substrate that is expected to facilitate the construction of high-density recording and stimulating arrays. Approach. Individual carbon fibers were aligned using an alignment tool that was 3D-printed with sub-micron resolution using direct laser writing. Indium deposition on the carbon fibers, followed by low-temperature microsoldering, provided a robust and reliable method of electrical connection to the polyimide interconnect. Main results. Spontaneous multiunit activity and stimulation-evoked compound responses with SNR >10 and >120, respectively, were recorded from a small (125 µm) peripheral nerve. We also improved the typically poor charge injection capacity of small diameter carbon fibers by electrodepositing 100 nm-thick iridium oxide films, making the carbon fiber arrays usable for electrical stimulation as well as recording. Significance. Our innovations in fabrication technique pave the way for further miniaturization of carbon fiber ultra-microelectrode arrays. We believe these advances to be key steps to enable a shift from labor intensive, manual assembly to a more automated manufacturing process.

OBJECTIVE Spinal cord injury causes a drastic loss of motor, sensory and autonomic function. The goal of this project was to investigate the use of intraspinal microstimulation (ISMS) for producing long distances of walking over ground. ISMS is an electrical stimulation method developed for restoring motor function by activating spinal networks below the level of an injury. It produces movements of the legs by stimulating the ventral horn of the lumbar enlargement using fine penetrating electrodes (≤50 μm diameter). APPROACH In each of five adult cats (4.2-5.5 kg), ISMS was applied through 16 electrodes implanted with tips targeting lamina IX in the ventral horn bilaterally. A desktop system implemented a physiologically-based control strategy that delivered different stimulation patterns through groups of electrodes to evoke walking movements with appropriate limb kinematics and forces corresponding to swing and stance. Each cat walked over an instrumented 2.9 m walkway and limb kinematics and forces were recorded. MAIN RESULTS Both propulsive and supportive forces were required for over-ground walking. Cumulative walking distances ranging from 609 to 835 m (longest tested) were achieved in three animals. In these three cats, the mean peak supportive force was 3.5 ± 0.6 N corresponding to full-weight-support of the hind legs, while the angular range of the hip, knee, and ankle joints were 23.1 ± 2.0°, 29.1 ± 0.2°, and 60.3 ± 5.2°, respectively. To further demonstrate the viability of ISMS for future clinical use, a prototype implantable module was successfully implemented in a subset of trials and produced comparable walking performance. SIGNIFICANCE By activating inherent locomotor networks within the lumbosacral spinal cord, ISMS was capable of producing bilaterally coordinated and functional over-ground walking with current amplitudes

Neural pathways can be artificially activated through the use of electrical stimulation. For individuals with a spinal cord injury, intraspinal microstimulation, using electrical currents on the order of 125 μ A, can produce muscle contractions and joint torques in the lower extremities suitable for restoring walking. The work presented here demonstrates an integrated circuit implementing a state-based control strategy where sensory feedback and intrinsic feed forward control shape the stimulation waveforms produced on-chip. Fabricated in a 0.5 μ m process, the device was successfully used in vivo to produce walking movements in a model of spinal cord injury. This work represents progress towards an implantable solution to be used for restoring walking in individuals with spinal cord injuries.

Objective. The vision of bioelectronic medicine is to treat disease by modulating the signaling of visceral nerves near various end organs. In small animal models, the nerves of interest can have small diameters and limited surgical access. New high-resolution methods for building nerve interfaces are desirable. In this study, we present a novel nerve interface and demonstrate its use for stimulation and recording in small nerves. Approach. We design and fabricate micro-scale electrode-laden nanoclips capable of interfacing with nerves as small as 50 µm in diameter. The nanoclips are fabricated using a direct laser writing technique with a resolution of 200 nm. The resolution of the printing process allows for incorporation of a number of innovations such as trapdoors to secure the device to the nerve, and quick-release mounts that facilitate keyhole surgery, obviating the need for forceps. The nanoclip can be built around various electrode materials; here we use carbon nanotube fibers for minimally invasive tethering. Main results. We present data from stimulation-evoked responses of the tracheal syringeal (hypoglossal) nerve of the zebra finch, as well as quantification of nerve functionality at various time points post implant, demonstrating that the nanoclip is compatible with healthy nerve activity over sub-chronic timescales. Significance. Our nerve interface addresses key challenges in interfacing with small nerves in the peripheral nervous system. Its small size, ability to remain on the nerve over sub-chronic timescales, and ease of implantation, make it a promising tool for future use in the treatment of disease.

A proposed Locomotion Processing Unit (LPU) is described for generating stimulation patterns for restoring walking in individuals with spinal cord injury (SCI). The LPU operates using sensory and timing based control providing feed forward and feedback information. By breaking down different components of locomotion into states, the LPU activates different muscle groups, or synergies, to recreate the desired functional movements. The LPU circuitry was simulated and compared against another controller designed to restore locomotion in an anesthetized cat to validate its performance.

Objective. The goal of this study was to decode sensory information from the dorsal root ganglia (DRG) in real time, and to use this information to adapt the control of unilateral stepping with a state-based control algorithm consisting of both feed-forward and feedback components. Approach. In five anesthetized cats, hind limb stepping on a walkway or treadmill was produced by patterned electrical stimulation of the spinal cord through implanted microwire arrays, while neuronal activity was recorded from the DRG. Different parameters, including distance and tilt of the vector between hip and limb endpoint, integrated gyroscope and ground reaction force were modelled from recorded neural firing rates. These models were then used for closed-loop feedback. Main results. Overall, firing-rate-based predictions of kinematic sensors (limb endpoint, integrated gyroscope) were the most accurate with variance accounted for >60% on average. Force prediction had the lowest prediction accuracy (48 ± 13%) but produced the greatest percentage of successful rule activations (96.3%) for stepping under closed-loop feedback control. The prediction of all sensor modalities degraded over time, with the exception of tilt. Significance. Sensory feedback from moving limbs would be a desirable component of any neuroprosthetic device designed to restore walking in people after a spinal cord injury. This study provides a proof-of-principle that real-time feedback from the DRG is possible and could form part of a fully implantable neuroprosthetic device with further development.

The biological central pattern generator (CPG) integrates open and closed loop control to produce over-ground walking. The goal of this study was to develop a physiologically based algorithm capable of mimicking the biological system to control multiple joints in the lower extremities for producing over-ground walking. The algorithm used state-based models of the step cycle each of which produced different stimulation patterns. Two configurations were implemented to restore over-ground walking in five adult anaesthetized cats using intramuscular stimulation (IMS) of the main hip, knee and ankle flexor and extensor muscles in the hind limbs. An open loop controller relied only on intrinsic timing while a hybrid-CPG controller added sensory feedback from force plates (representing limb loading), and accelerometers and gyroscopes (representing limb position). Stimulation applied to hind limb muscles caused extension or flexion in the hips, knees and ankles. A total of 113 walking trials were obtained across all experiments. Of these, 74 were successful in which the cats traversed 75% of the 3.5 m over-ground walkway. In these trials, the average peak step length decreased from 24.9 ± 8.4 to 21.8 ± 7.5 (normalized units) and the median number of steps per trial increased from 7 (Q1 = 6, Q3 = 9) to 9 (8, 11) with the hybrid-CPG controller. Moreover, within these trials, the hybrid-CPG controller produced more successful steps (step length ≤ 20 cm; ground reaction force ≥ 12.5% body weight) than the open loop controller: 372 of 544 steps (68%) versus 65 of 134 steps (49%), respectively. This supports our previous preliminary findings, and affirms that physiologically based hybrid-CPG approaches produce more successful stepping than open loop controllers. The algorithm provides the foundation for a neural prosthetic controller and a framework to implement more detailed control of locomotion in the future.