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.

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.

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.

Applications of Graphene and Graphene Oxide in Smart Drug/Gene Delivery: Is the World Still Flat?

Graphene, a wonder material, has made far-reaching developments in many different fields such as materials science, electronics, condensed physics, quantum physics, energy systems, etc. Since its discovery in 2004, extensive studies have been done for understanding its physical and chemical properties. Owing to its unique characteristics, it has rapidly became a potential candidate for nano-bio researchers to explore its usage in biomedical applications. In the last decade, remarkable efforts have been devoted to investigating the biomedical utilization of graphene and graphene-based materials, especially in smart drug and gene delivery as well as cancer therapy. Inspired by a great number of successful graphene-based materials integrations into the biomedical area, here we summarize the most recent developments made about graphene applications in biomedicine. In this paper, we review the up-to-date advances of graphene-based materials in drug delivery applications, specifically targeted drug/ gene delivery, delivery of antitumor drugs, controlled and stimuli-responsive drug release, photodynamic therapy applications and optical imaging and theranostics, as well as investigating the future trends and succeeding challenges in this topic to provide an outlook for future researches.

Bacterial components as naturally inspired nano-carriers for drug/gene delivery and immunization: Set the bugs to work?

Drug delivery is a rapidly growing area of research motivated by the nanotechnology revolution, the ideal of personalized medicine, and the desire to reduce the side effects of toxic anti-cancer drugs. Amongst a bewildering array of different nanostructures and nanocarriers, those examples that are fundamentally bio-inspired and derived from natural sources are particularly preferred. Delivery of vaccines is also an active area of research in this field. Bacterial cells and their components that have been used for drug delivery, include the crystalline cell-surface layer known as "S-layer", bacterial ghosts, bacterial outer membrane vesicles, and bacterial products or derivatives (e.g. spores, polymers, and magnetic nanoparticles). Considering the origin of these components from potentially pathogenic microorganisms, it is not surprising that they have been applied for vaccines and immunization. The present review critically summarizes their applications focusing on their advantages for delivery of drugs, genes, and vaccines.

Stimulus-responsive liposomes as smart nanoplatforms for drug delivery applications.

Liposomes are known to be promising nanoparticles (NPs) for drug delivery applications. Among different types of self-assembled NPs, liposomes stand out for their non-toxic nature, and their possession of dual hydrophilic-hydrophobic domains. Advantages of liposomes include the ability to solubilize hydrophobic drugs, the ability to incorporate different hydrophilic and lipophilic drugs at the same time, lessening the exposure of host organs to potentially toxic drugs and allowing modification of the surface by a variety of different chemical groups. This modification of the surface, or of the individual constituents, may be used to achieve two important goals. Firstly, ligands for active targeting can be attached that are recognized by cognate receptors over-expressed on the target cells of tissues. Secondly, modification can be used to impart a stimulus-responsive or "smart" character to the liposomes, whereby the cargo is released on demand only when certain internal stimuli (pH, reducing agents, specific enzymes) or external stimuli (light, magnetic field or ultrasound) are present. Here, we review the field of smart liposomes for drug delivery applications.