Safety of mapping the motor networks in the spinal cord using penetrating microelectrodes in Yucatan minipigs.

OBJECTIVE: The goal of this study was to assess the safety of mapping spinal cord locomotor networks using penetrating stimulation microelectrodes in Yucatan minipigs (YMPs) as a clinically translational animal model. METHODS: Eleven YMPs were trained to walk up and down a straight line. Motion capture was performed, and electromyographic (EMG) activity of hindlimb muscles was recorded during overground walking. The YMPs underwent a laminectomy and durotomy to expose the lumbar spinal cord. Using an ultrasound-guided stereotaxic frame, microelectrodes were inserted into the spinal cord in 8 animals. Pial cuts were made to prevent tissue dimpling before microelectrode insertion. Different locations within the lumbar enlargement were electrically stimulated to map the locomotor networks. The remaining 3 YMPs served as sham controls, receiving the laminectomy, durotomy, and pial cuts but not microelectrode insertion. The Porcine Thoracic Injury Behavioral Scale (PTIBS) and hindlimb reflex assessment results were recorded for 4 weeks postoperatively. Overground gait kinematics and hindlimb EMG activity were recorded again at weeks 3 and 4 postoperatively and compared with preoperative measures. The animals were euthanized at the end of week 4, and the lumbar spinal cords were extracted and preserved for immunohistochemical analysis. RESULTS: All YMPs showed transient deficits in hindlimb function postoperatively. Except for 1 YMP in the experimental group, all animals regained normal ambulation and balance (PTIBS score 10) at the end of weeks 3 and 4. One animal in the experimental group showed gait and balance deficits by week 4 (PTIBS score 4). This animal was excluded from the kinematics and EMG analyses. Overground gait kinematic measures and EMG activity showed no significant (p > 0.05) differences between preoperative and postoperative values, and between the experimental and sham groups. Less than 5% of electrode tracks were visible in the tissue analysis of the animals in the experimental group. There was no statistically significant difference in damage caused by pial cuts between the experimental and sham groups. Tissue damage due to the pial cuts was more frequently observed in immunohistochemical analyses than microelectrode tracks. CONCLUSIONS: These findings suggest that mapping spinal locomotor networks in porcine models can be performed safely, without lasting damage to the spinal cord.

Development of wafer-scale multifunctional nanophotonic neural probes for brain activity mapping.

Optical techniques, such as optogenetic stimulation and functional fluorescence imaging, have been revolutionary for neuroscience by enabling neural circuit analysis with cell-type specificity. To probe deep brain regions, implantable light sources are crucial. Silicon photonics, commonly used for data communications, shows great promise in creating implantable devices with complex optical systems in a compact form factor compatible with high volume manufacturing practices. This article reviews recent developments of wafer-scale multifunctional nanophotonic neural probes. The probes can be realized on 200 or 300 mm wafers in commercial foundries and integrate light emitters for photostimulation, microelectrodes for electrophysiological recording, and microfluidic channels for chemical delivery and sampling. By integrating active optical devices to the probes, denser emitter arrays, enhanced on-chip biosensing, and increased ease of use may be realized. Silicon photonics technology makes possible highly versatile implantable neural probes that can transform neuroscience experiments.

In vitro biocompatibility evaluation of functional electrically stimulating microelectrodes on primary glia.

Neural interfacing devices interact with the central nervous system to alleviate functional deficits arising from disease or injury. This often entails the use of invasive microelectrode implants that elicit inflammatory responses from glial cells and leads to loss of device function. Previous work focused on improving implant biocompatibility by modifying electrode composition; here, we investigated the direct effects of electrical stimulation on glial cells at the electrode interface. A high-throughput in vitro system that assesses primary glial cell response to biphasic stimulation waveforms at 0 mA, 0.15 mA, and 1.5 mA was developed and optimized. Primary mixed glial cell cultures were generated from heterozygous CX3CR-1+/EGFP mice, electrically stimulated for 4 h/day over 3 days using 75 µm platinum-iridium microelectrodes, and biomarker immunofluorescence was measured. Electrodes were then imaged on a scanning electron microscope to assess sustained electrode damage. Fluorescence and electron microscopy analyses suggest varying degrees of localized responses for each biomarker assayed (Hoescht, EGFP, GFAP, and IL-1ß), a result that expands on comparable in vivo models. This system allows for the comparison of a breadth of electrical stimulation parameters, and opens another avenue through which neural interfacing device developers can improve biocompatibility and longevity of electrodes in tissue.

Laser-Microfabricated Polymer Multielectrodes for Intraspinal Microstimulation.

OBJECTIVE: The overall goal of this study was to design, fabricate, and characterize a new polymer-based multielectrode for the spinal cord for the application of intraspinal microstimulation (ISMS). METHODS: Three-channel multielectrodes were fabricated from modified poly(dimethylsiloxane) (PDMS) and platinum-iridium (Pt-Ir) foil using nanosecond laser microfabrication techniques. These devices were compared against traditional 50 µm diameter Pt-Ir microwire electrodes mechanically and electrochemically in bench environments, and were assessed electrochemically and functionally in vivo in a domestic pig model. RESULTS: Polymer-based multielectrodes were significantly more flexible than microwire electrodes (p < 0.05) and had greater charge storage capacities in phosphate buffered saline (p < 0.05). In a domestic pig model, multielectrodes had significantly greater charge injection limits than microwire electrodes (p < 0.05). When stimulating within the quadriceps motor pool in the spinal cord, multielectrodes generated strong knee extensor joint torques of up to 4.4 ± 0.3 Nm and were able to extend the knee by up to 26 ± 1°. However, histological analyses showed that polymer-based multielectrodes, implanted with half-needle insertion aids, produced greater acute tissue damage compared to microwire electrodes (p < 0.05). Alternative insertion methods for these flexible electrodes should be explored to reduce acute tissue damage. CONCLUSION: The PDMS-based three-channel multielectrodes demonstrated improved flexibility and charge injection capabilities over traditional microwire electrodes, and were able to produce functional responses in vivo. SIGNIFICANCE: Polymer-based multielectrodes demonstrate improved functionality over microwire electrodes while remaining more flexible than silicon multielectrode designs. These features may in the future permit polymer-based multielectrodes to implement ISMS with greater efficacy and biocompatibility compared to traditional technologies.

GABA facilitates spike propagation through branch points of sensory axons in the spinal cord.

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons directly with optogenetics or indirectly by cutaneous stimulation actually facilitates sensory feedback to motor neurons in rodents and humans. GABAA receptors located at or near nodes of Ranvier of sensory axons cause this facilitation by preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals and so cannot inhibit transmitter release onto motor neurons, unlike GABAB receptors that cause presynaptic inhibition. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other central nervous system axons.

Overground gait kinematics and muscle activation patterns in the Yucatan mini pig.

Objective.The objectives of this study were to assess gait biomechanics and the effect of overground walking speed on gait parameters, kinematics, and electromyographic (EMG) activity in the hindlimb muscles of Yucatan minipigs (YMPs).Approach.Nine neurologically-intact, adult YMPs were trained to walk overground in a straight line. Whole-body kinematics and EMG activity of hindlimb muscles were recorded and analyzed at six different speed ranges (0.4-0.59, 0.6-0.79, 0.8-0.99, 1.0-1.19, 1.2-1.39, and 1.4-1.6 m s-1). A MATLAB program was developed to detect strides and gait events automatically from motion-captured data. The kinematics and EMG activity were analyzed for each stride based on the detected events.Main results.Significant decreases in stride duration, stance and swing times and an increase in stride length were observed with increasing speed. A transition in gait pattern occurred at the 1.0 m s-1walking speed. Significant increases in the range of motion of the knee and ankle joints were observed at higher speeds. Also, the points of minimum and maximum joint angles occurred earlier in the gait cycle as the walking speed increased. The onset of EMG activity in the biceps femoris muscle occurred significantly earlier in the gait cycle with increasing speed.Significance.YMPs are becoming frequently used as large animal models for preclinical testing and translation of novel interventions to humans. A comprehensive characterization of overground walking in neurologically-intact YMPs is provided in this study. These normative measures set the basis against which the effects of future interventions on locomotor capacity in YMPs can be compared.

Pavlovian control of intraspinal microstimulation to produce over-ground walking.

OBJECTIVE: Neuromodulation technologies are increasingly used for improving function after neural injury. To achieve a symbiotic relationship between device and user, the device must augment remaining function, and independently adapt to day-to-day changes in function. The goal of this study was to develop predictive control strategies to produce over-ground walking in a model of hemisection spinal cord injury (SCI) using intraspinal microstimulation (ISMS). APPROACH: Eight cats were anaesthetized and placed in a sling over a walkway. The residual function of a hemisection SCI was mimicked by manually moving one hind-limb through the walking cycle. ISMS targeted motor networks in the lumbosacral enlargement to activate muscles in the other, presumably 'paralyzed' limb, using low levels of current (<130 µA). Four people took turns to move the 'intact' limb, generating four different walking styles. Two control strategies, which used ground reaction force and angular velocity information about the manually moved 'intact' limb to control the timing of the transitions of the 'paralyzed' limb through the step cycle, were compared. The first strategy used thresholds on the raw sensor values to initiate transitions. The second strategy used reinforcement learning and Pavlovian control to learn predictions about the sensor values. Thresholds on the predictions were then used to initiate transitions. MAIN RESULTS: Both control strategies were able to produce alternating, over-ground walking. Transitions based on raw sensor values required manual tuning of thresholds for each person to produce walking, whereas Pavlovian control did not. Learning occurred quickly during walking: predictions of the sensor signals were learned rapidly, initiating correct transitions after ≤4 steps. Pavlovian control was resilient to different walking styles and different cats, and recovered from induced mistakes during walking. SIGNIFICANCE: This work demonstrates, for the first time, that Pavlovian control can augment remaining function and facilitate personalized walking with minimal tuning requirements.