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The biomechanics of peripheral nerves are determined by the blood-nerve barrier (BNB), together with the epineural barrier, extracellular matrix, and axonal composition, which maintain structural and functional stability. These elements are often ignored in the fabrication of penetrating devices, and the implant process is traumatic due to the mechanical distress, compromising the function of neuroprosthesis for sensory-motor restoration in amputees. Miniaturization of penetrating interfaces offers the unique opportunity of decoding individual nerve fibers associated to specific functions, however, a main issue for their implant is the lack of high-precision standardization of insertion forces. Current automatized electromechanical force sensors are available; however, their sensitivity and range amplitude are limited (i.e. mN), and have been tested onlyin-vitro. We previously developed a high-precision bi-directional micro-electromechanical force sensor, with a closed-loop mechanism (MEMS-CLFS), that while measuring with high-precision (−211.7μN to 211.5μN with a resolution of 4.74nN), can be used in alive animal. Our technology has an on-chip electrothermal displacement sensor with a shuttle beam displacement amplification mechanism, for large range and high-frequency resolution (dynamic range of 92.9 dB), which eliminates the adverse effect of flexural nonlinearity measurements, observed with other systems, and reduces the mechanical impact on delicate biological tissue. In this work, we use the MEMS-CLFS forin-vivobidirectional measurement of biomechanics in somatic and autonomic nerves. Furthermore we define the mechanical implications of irrigation and collagen VI in the BNB, which is different for both autonomic and somatic nerves (∼ 8.5-8.6 fold density of collagen VI and vasculature CD31+ in the VN vs ScN). This study allowed us to create a mathematical approach to predict insertion forces. Our data highlights the necessity of nerve-customization forces to prevent injury when implanting interfaces, and describes a high precision MEMS technology and mathematical model for their measurements.

Stress urinary incontinence (SUI) is characterized by the involuntary loss of urine due to increased intra-abdominal pressure during coughing, sneezing, or exercising. SUI affects 20-40% of the female population and is exacerbated by aging. Severe SUI is commonly treated with surgical implantation of an autologous or a synthetic sling underneath the urethra for support. These slings, however, are static, and their tension cannot be non-invasively adjusted, if needed, after implantation. This study reports the fabrication of a novel device based on liquid crystal elastomers (LCEs) capable of changing shape in response to temperature increase induced by transcutaneous IR light. The shape change of the LCE-based device was characterized in a scar tissue phantom model. An in vitro urinary tract model was designed to study the efficacy of the LCE-based device to support continence and adjust sling tension with IR illumination. Finally, the device was acutely implanted and tested for induced tension changes in female multiparous New Zealand white rabbits. The LCE device achieved 5.6% ± 1.1% actuation when embedded in an agar gel with an elastic modulus of 100 kPa. The corresponding device temperature was 44.9 °C ± 0.4 °C, and the surrounding agar temperature stayed at 42.1 °C ± 0.4 °C. Leaking time in the in vitro urinary tract model significantly decreased (p 0.0001) when an LCE-based cuff was sutured around the model urethra from 5.2min ± 1min to 2min ±0.5min when the cuff was illuminated with IR light. Normalized leak point force (LPF) increased significantly (p = 0.01) with the implantation of an LCE-CB cuff around the bladder neck of multiparous rabbits. It decreased significantly (p = 0.023) when the device was actuated via IR light illumination. These results demonstrate that LCE material could be used to fabricate a dynamic device for treating SUI in women.

Pelvic floor muscle stretch injury during pregnancy and birth is associated with the incidence of stress urinary incontinence (SUI), a condition that affects 30–60% of the female population and is characterized by involuntary urine leakage during physical activity, further exacerbated by aging. Aging and multiparous rabbits suffer pelvic nerve and muscle damage, resulting in alterations in pelvic floor muscular contraction and low urethral pressure, resembling SUI. However, the extent of nerve injury is not fully understood. Here, we used electron microscopy analysis of pelvic and perineal nerves in multiparous rabbits to describe the extent of stretch nerve injury based on axon count, axon size, myelin-to-axon ratio, and elliptical ratio. Compared to young nulliparous controls, mid-age multiparous animals showed an increase in the density of unmyelinated axons and in myelin thickness in both nerves, albeit more significant in the bulbospongiosus nerve. This revealed a partial but sustained damage to these nerves, and the presence of some regenerated axons. Additionally, we tested whether electrical stimulation of the bulbospongiosus nerve would induce muscle contraction and urethral closure. Using a miniature wireless stimulator implanted on this perineal nerve in young nulliparous and middle age multiparous female rabbits, we confirmed that these partially damaged nerves can be acutely depolarized, either at low (2–5 Hz) or medium (10–20 Hz) frequencies, to induce a proportional increase in urethral pressure. Evaluation of micturition volume in the mid-age multiparous animals after perineal nerve stimulation, effectively reversed a baseline deficit, increasing it 2-fold (p = 0.02). These results support the notion that selective neuromodulation of pelvic floor muscles might serve as a potential treatment for SUI.

Purpose of Review Pain presents a unique challenge due to the complexity of the biological pathways involved in the pain perception, the growing concern regarding the use of opioid analgesics, and the limited availability of optimal treatment options. The use of biomaterials and regenerative medicine in pain management is being actively explored and showing exciting progress in improving the efficacy of conventional pharmacotherapy and as novel non-pharmacological therapy for chronic pain caused by degenerative diseases. In this paper we review current clinical applications, and promising research in the use of biomaterials and regenerative medicine in pain management. Recent Findings Regenerative therapies have been developed to repair damaged tissues in back, joint, and shoulder that lead to chronic and inflammatory pain. Novel regenerative biomaterials have been designed to incorporate biochemical and physical pro-regenerative cues that augment the efficacy of regenerative therapies. New biomaterials improve target localization with improved tunability for controlled drug delivery, and injectable scaffolds enhance the efficacy of regenerative therapies through improving cellular migration. Advanced biomaterial carrier systems have been developed for sustained and targeted delivery of analgesic agents to specific tissues and organs, showing improved treatment efficacy, extended duration of action, and reduced dosage. Targeting endosomal receptors by nanoparticles has shown promising anti-nociception effects. Biomaterial scavengers are designed to remove proinflammatory reactive oxygen species that trigger nociceptors and cause pain hypersensitivity, providing a proactive approach for pain management. Summary Pharmacotherapy remains the method of choice for pain management; however, conventional analgesic agents are associated with adverse effects. The relatively short duration of action when applied as free drug limited their efficacy in postoperative and chronic pain treatment. The application of biomaterials in pain management is a promising strategy to improve the efficacy of current pharmacotherapy through sustained and targeted delivery of analgesic agents. Regenerative medicine strategies target the damaged tissue and provide non-pharmacological alternatives to manage chronic and inflammatory pain. In the future, the successful development of regenerative therapies that completely repair damaged tissues will provide a more optimal alternative for the treatment of chronic pain caused. Future studies will leverage on the increasing understanding of the molecular mechanisms governing pain perception and transmission, injury response and tissue regeneration, and the development of new biomaterials and tissue regenerative methods.

Neural interfacing nerve fascicles along the splenic neurovascular plexus (SNVP) is needed to better understand the spleen physiology, and for selective neuromodulation of this major organ. However, their small size and anatomical location have proven to be a significant challenge. Here, we use a reduced liquid crystalline graphene oxide (rGO) fiber coated with platinum (Pt) as a super-flexible suture-like electrode to interface multiple SNVP. The Pt-rGO fibers work as a handover knot electrodes over the small SNVP, allowing sensitive recording from four splenic nerve terminal branches (SN 1–4), to uncover differential activity and axon composition among them. Here, the asymmetric defasciculation of the SN branches is revealed by electron microscopy, and the functional compartmentalization in spleen innervation is evidenced in response to hypoxia and pharmacological modulation of mean arterial pressure. We demonstrate that electrical stimulation of cervical and sub-diaphragmatic vagus nerve (VN), evokes activity in a subset of SN terminal branches, providing evidence for a direct VN control over the spleen. This notion is supported by adenoviral tract-tracing of SN branches, revealing an unconventional direct brain-spleen projection. High-performance Pt-rGO fiber electrodes, may be used for the fine neural modulation of other small neurovascular plexus at the point of entry of major organs as a bioelectronic medical alternative., Gonzalez-Gonzalez et al. use high-performance platinized graphene fiber electrodes to interface individual neurovascular plexus that innervate the spleen. Their approach provides evidence for distinct function of individual spleen terminal branches in organ function.

Hypertension affects nearly half of the US population but only 43% achieved blood pressure control with medication alone. Medical devices for hypertension include implantable lead electrodes that stimulate the carotid baroreceptors with promising results, albeit with significant adverse complications. To address these limitations, we have proposed the use of deep peroneal nerve stimulation (DPNS), which elicited a depressor response in anesthetized, breathing supported, spontaneously hypertensive rats (SHR). In this study, we further define the electrical stimulation parameters that optimize the DPNS depressor response, and demonstrated that increasing the pulse duration from 0.15 ms to 1ms, of 1.0 mA pulses at 2 Hz for 10 sec, significantly reduced the mean arterial pressure (MAP) by 8±4 mmHg (p

Neural interfacing nerve fascicles along the splenic neurovascular plexi (SNVP) is needed to better understand the spleen physiology, and for selective neuromodulation of this major organ. However, the small size and anatomical location have proven to be a significant challenge. Here, we use a reduced liquid crystalline graphene oxide (rGO) fiber coated with platinum (Pt) as a super-flexible suture-like electrode to interface multiple SNVP. The Pt-rGO fibers were used as handover knot electrodes over the small plexi, allowing sensitive recording from the splenic nerve (SN) terminal branches. Asymmetric defasciculation of the SN branches was revealed by electron microscopy, and the functional compartmentalization in spleen innervation was evidenced in response to hypoxia and pharmacological modulation of mean arterial pressure. We demonstrate that electrical stimulation of cervical and subdiaphragmatic vagus nerve (VN), evoked direct activity in a subset of SN terminal branches, providing evidence for a direct VN control over the spleen. This notion was supported by tract-tracing of SN branches, revealing an unconventional direct spleen-VN projection. High-performance Pt-rGO fiber electrodes, may be used for the fine neural modulation of other small neurovascular plexi at the point of entry of the organs as a bioelectronic medical alternative.

Obesity remains prevalent in the US. One potential treatment is vagus nerve stimulation (VNS), which activates the sensory afferents innervating the stomach that convey stomach volume and establish satiety. However, current VNS approaches and stimulus optimization could benefit from additional understanding of the underlying neural response to stomach distension. In this study, obesity-prone Sprague Dawley rats consumed a standard, high-carbohydrate, or high-fat diet for several months, leading to diet-induced obesity in the latter two groups. Under anesthesia, the neural activity in the vagus nerve was recorded with a penetrating microelectrode array while the stomach was distended with an implanted balloon. Vagal tone during distension was compared to baseline tone prior to distension. Responses were strongly correlated with stomach distension, but the sensitivity to distension was significantly lower in animals that had been fed the nonstandard diets. The results indicate that both high fat and high carbohydrate diets impair vagus activity.

The repair of nerve gap injuries longer than 3 cm is limited by the need to sacrifice donor tissue and the morbidity associated with the autograft gold standard, while decellularized grafts and biodegradable conduits are effective only in short nerve defects. The advantage of isogenic nerve implants seems to be the release of various growth factors by the denervated Schwann cells. We evaluated the effect of vascular endothelial growth factor, neurotrophins, and pleiotrophin (PTN) supplementation of multi-luminal conduits, in the repair of 3 and 4 cm nerve gaps in the rabbit peroneal nerve. In vitro screening revealed a synergistic regenerative effect of PTN with glial-derived neurotrophic factor (GDNF) in promoting sensory axon density, and motor axonal growth from spinal cord explants. In vivo, pleiotrophins were able to support nerve regrowth across a 3 cm gap. In the 4 cm lesions, PTN-GDNF had a modest effect in the number of axons distal to the implant, while increasing the mean axon diameter (1 ± 0.4; p ≤ 0.001) over PTN or GDNF alone (0.80 ± 0.2, 0.84 ± 0.5; respectively). Some regenerated axons reinnervated muscle targets as indicated by neuromuscular junction staining. However, many were wrapped in Remak bundles, suggesting a delay in axonal sorting, explaining the limited electrophysiological function of the reinnervated muscle, and the modest recovery in toe spreading in the PTN-GDNF repaired animals. These results support the use of synergistic neurotrophic/pleiotrophic growth factors in long gap repair and underscore the need for re-myelination strategies distal to the injury site. Statement of Significance Nerve injuries due to trauma or tumor resection often result in long gaps that are challenging to repair. The best clinical option demands the use of autologous grafts that are associated with serious side effects. Bioengineered nerves are considered a good alternative, particularly if supplemented with growth factors, but current options do not match the regenerative capacity of autografts. This study revealed the synergistic effect of neurotrophins and pleiotrophins designed to achieve a broad cellular regenerative effect, and that GDNF-PTN are able to mediated axonal growth and partial functional recovery in a 4 cm nerve gap injury, albeit delays in remyelination. This report underscores the need for defining an optimal growth factor support for biosynthetic nerve implants.

Following inflammation or injury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell- or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE2, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.

The ability to interface electronic materials with the peripheral nervous system is required for stimulation and monitoring of neural signals. Thus, the design and engineering of robust neural interfaces that maintain material-tissue contact in the presence of material or tissue micromotion offer the potential to conduct novel measurements and develop future therapies that require chronic interface with the peripheral nervous system. However, such remains an open challenge given the constraints of existing materials sets and manufacturing approaches for design and fabrication of neural interfaces. Here, we investigated the potential to leverage a rapid prototyping approach for the design and fabrication of nerve cuffs that contain supporting features to mechanically stabilize the interaction between cuff electrodes and peripheral nerve. A hybrid 3D printing and robotic-embedding (i.e., pick-and-place) system was used to design and fabricate silicone nerve cuffs (800 µm diameter) containing conforming platinum (Pt) electrodes. We demonstrate that the electrical impedance of the cuff electrodes can be reduced by deposition of the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on cuff electrodes via a post-processing electropolymerization technique. The computer-aided design and manufacturing approach was also used to design and integrate supporting features to the cuff that mechanically stabilize the interface between the cuff electrodes and the peripheral nerve. Both ‘self-locking’ and suture-assisted locking mechanisms are demonstrated based on the principle of making geometric alterations to the cuff opening via 3D printing. Ultimately, this work shows 3D printing offers considerable opportunity to integrate supporting features, and potentially even novel electronic materials, into nerve cuffs that can support the design and engineering of next generation neural interfaces.

Neural interfaces are designed to decode motor intent and evoke sensory precepts in amputees. In peripheral nerves, recording movement intent is challenging because motor axons are only a small fraction compared to sensory fibers and are heterogeneously mixed particularly at proximal levels. We previously reported that pain and myelinated axons regenerating through a Y-shaped nerve guide with sealed ends, can be modulated by luminar release of nerve growth factor (NGF) and neurotrophin-3 (NT-3), respectively. Here, we evaluate the differential potency of NGF, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), pleiotrophin (PTN), and NT-3 in asymmetrically guiding the regeneration of sensory and motor neurons. We report that, in the absence of distal target organs, molecular guidance cues can mediate the growth of electrically conductive fascicles with normal microanatomy. Compared to Y-tube compartments with bovine serum albumin (BSA), GDNF and NGF increased the motor and sensory axon content, respectively. In addition, the sensory to motor ratio was significantly increased by PTN (12.7:1) when compared to a BDNF + GDNF choice. The differential content of motor and sensory axons modulated by selective guidance cues may provide a strategy to better define axon types in peripheral nerve interfaces.

Corrective forces during spine deformity surgery, including distraction, impart significant stresses to the spinal cord that may result in permanent injury. Intraoperative neuromonitoring is commonly used by surgeons to recognize possible damage to the spinal cord in cases of evident traumatic or vascular damage to the spinal cord. However, mild insult to the spinal cord that does not result in obvious trauma or electrophysiological changes present a major clinical challenge as the mechanisms of this type of spinal cord injury (SCI) remain largely unknown, and thus preventive strategies are lacking. We used a sustained bidirectional spinal distraction animal model to determine the role of stretch-induced hypoxia in mild SCI. Direct measurement of intraparenchymal oxygen revealed an immediate decrease in partial pressure (47.08 ± 5.79% pO2) distal to the injury site following a 5-mm distraction. This hypoxic insult induced mitochondrial dysfunction as evidenced by an acute increase (216%) in protein oxidation 30 min post-injury, as well as a 37% decrease in perikaryal size and a 42% decrease in nuclear area (pyknosis) in ventral motor neurons at the injury site. These results indicate that hypoxic events during mild spine distraction may lead to cellular metabolic impairments and permanent functional deficits. The development of strategies targeting the prevention of hypoxic injury during spine distraction may be useful in protecting the cellular metabolic damage that may occur during spine surgery in the absence of overt mechanical or vascular SCI.

The majority of available systems for nerve stimulation use circumferential stimulation electrodes inside an insulating cuff, which produce largely uniform current density within the nerve. Flat stimulation electrodes that contact only one side of the nerve may provide advantages including simpler implantation, ease of production, and more resistance to mechanical failure. However, it is possible that the flat configuration will yield inefficient fiber recruitment due to a less uniform current distribution within the nerve. Here we tested the hypothesis that flat electrodes will require higher current amplitude to achieve effective stimulation than circumferential designs. Computational modeling and in vivo experiments were performed to evaluate fiber recruitment in different nerves and different species using a variety of electrode designs. Initial results demonstrated similar fiber recruitment in the rat vagus and sciatic nerves with a standard circumferential cuff electrode and a cuff electrode modified to approximate a flat configuration. Follow up experiments comparing true flat electrodes to circumferential electrodes on the rabbit sciatic nerve confirmed that fiber recruitment was equivalent between the two designs. These findings demonstrate that flat electrodes represent a viable design for nerve stimulation that may provide advantages over the current circumferential designs for applications in which the goal is uniform activation of the nerve.

Nerve damage can cause chronic, debilitating problems including loss of motor control and paresthesia, and generates maladaptive neuroplasticity as central networks attempt to compensate for the loss of peripheral connectivity. However, it remains unclear if this is a critical feature responsible for the expression of symptoms. Here, we use brief bursts of closed-loop vagus nerve stimulation (CL-VNS) delivered during rehabilitation to reverse the aberrant central plasticity resulting from forelimb nerve transection. CL-VNS therapy drives extensive synaptic reorganization in central networks paralleled by improved sensorimotor recovery without any observable changes in the nerve or muscle. Depleting cortical acetylcholine blocks the plasticity-enhancing effects of CL-VNS and consequently eliminates recovery, indicating a critical role for brain circuits in recovery. These findings demonstrate that manipulations to enhance central plasticity can improve sensorimotor recovery and define CL-VNS as a readily translatable therapy to restore function after nerve damage., Peripheral nerve damage generates maladaptive neuroplasticity as central networks attempt to compensate for the loss of peripheral connectivity. Here, the authors reverse the aberrant plasticity via vagus nerve stimulation to elicit synaptic reorganization and to improve sensorimotor recovery.

Neuromuscular junctions (NMJ) are specialized synapses that link motor neurons with muscle fibers. These sites are fundamental to human muscle activity, controlling swallowing and breathing amongst many other vital functions. Study of this synapse formation is an essential area in neuroscience; the understanding of how neurons interact and control their targets during development and regeneration are fundamental questions. Existing data reveals that during initial stages of development neurons target and form synapses driven by biophysical and biochemical cues, and during later stages they require electrical activity to develop their functional interactions. The aim of this study was to investigate the effect of exogenous electrical stimulation (ES) electrodes directly in contact with cells, on the number and size of acetylcholine receptor (AChR) clusters available for NMJ formation. We used a novel in vitro model that utilizes a flexible electrical stimulation system and allows the systematic testing of several stimulation parameters simultaneously as well as the use of alternative electrode materials such as conductive polymers to deliver the stimulation. Functionality of NMJs under our co-culture conditions was demonstrated by monitoring changes in the responses of primary myoblasts to chemical stimulants that specifically target neuronal signaling. Our results suggest that biphasic electrical stimulation at 250 Hz, 100 μs pulse width and current density of 1 mA/cm2 for 8 h, applied via either gold-coated mylar or the conductive polymer PPy, significantly increased the number and size of AChRs clusters available for NMJ formation. This study supports the beneficial use of direct electrical stimulation as a strategic therapy for neuromuscular disorders. Statement of Significance The beneficial effects of electrical stimulation (ES) on human cells in vitro and in vivo have long been known. Although the effects of stimulation are clear and the therapeutic benefits are known, no uniform parameters exist with regard to the duration, frequency and amplitude of the ES. To this end, we are answering several important questions on the parameters for ES of nerve and muscle monocultures and co-cultures by probing the effects on the enhancement of acetylcholine receptors (AChR) clustering available for neuromuscular junction formation using a conductive platform. This work opens the possibility to combine electrical stimulus delivered via conductive polymer substrates, from which biomolecules could also be delivered, providing opportunities to further enhance the therapeutic effect.

Intraoperative neurophysiological monitoring (IONM) is utilized to minimize neurological morbidity during spine surgery. Transcranial motor evoked potentials (TcMEPs) are principal IONM signals in which the motor cortex of the subject is stimulated with electrical pulses and the evoked potentials are recorded from the muscles of interest. Currently available monitoring systems require the connection of 40-60 lengthy lead wires to the patient. These wires contribute to a crowded and cluttered surgical environment, and limit the maneuverability of the surgical team. In this work, it was demonstrated that the cumbersome wired system is vulnerable to electromagnetic interference (EMI) produced by operating room (OR) equipment. It was hypothesized that eliminating the lengthy recording wires can remove the EMI induced in the IONM signals. Hence, a wireless system to acquire TcMEPs was developed and validated through bench-top and animal experiments. Side-by-side TcMEPs acquisition from the wired and wireless systems in animal experiments under controlled conditions (absence of EMI from OR equipment) showed comparable magnitudes and waveforms, thus demonstrating the fidelity in the signal acquisition of the wireless solution. The robustness of the wireless system to minimize EMI was compared with a wired-system under identical conditions. Unlike the wired-system, the wireless system was not influenced by the electromagnetic waves from the C-Arm X-ray machine and temperature management system in the OR.

The majority of available systems for vagus nerve stimulation use helical stimulation electrodes, which cover the majority of the circumference of the nerve and produce largely uniform current density within the nerve. Flat stimulation electrodes that contact only one side of the nerve may provide advantages, including ease of fabrication. However, it is possible that the flat configuration will yield inefficient fiber recruitment due to a less uniform current distribution within the nerve. Here we tested the hypothesis that flat electrodes will require higher current amplitude to activate all large-diameter fibers throughout the whole cross-section of a nerve than circumferential designs. Computational modeling and in vivo experiments were performed to evaluate fiber recruitment in different nerves and different species using a variety of electrode designs. Initial results demonstrated similar fiber recruitment in the rat vagus and sciatic nerves with a standard circumferential cuff electrode and a cuff electrode modified to approximate a flat configuration. Follow up experiments comparing true flat electrodes to circumferential electrodes on the rabbit sciatic nerve confirmed that fiber recruitment was equivalent between the two designs. These findings demonstrate that flat electrodes represent a viable design for nerve stimulation that may provide advantages over the current circumferential designs for applications in which the goal is uniform activation of all fascicles within the nerve.

Silicone nerve cuff electrodes are commonly implanted on relatively large and accessible somatic nerves as peripheral neural interfaces. While these cuff electrodes are soft (1–50 MPa), their self-closing mechanism requires of thick walls (200–600 µm), which in turn contribute to fibrotic tissue growth around and inside the device, compromising the neural interface. We report the use of thiol-ene/acrylate shape memory polymer (SMP) for the fabrication of thin film multi-electrode softening cuffs (MSC). We fabricated multi-size MSC with eight titanium nitride (TiN) electrodes ranging from 1.35 to 13.95 × 10−4 cm2 (1–3 kΩ) and eight smaller gold (Au) electrodes (3.3 × 10−5 cm2; 750 kΩ), that soften at physiological conditions to a modulus of 550 MPa. While the SMP material is not as soft as silicone, the flexural forces of the SMP cuff are about 70–700 times lower in the MSC devices due to the 30 μm thick film compared to the 600 μm thick walls of the silicone cuffs. We demonstrated the efficacy of the MSC to record neural signals from rat sciatic and pelvic nerves (1000 µm and 200 µm diameter, respectively), and the selective fascicular stimulation by current steering. When implanted side-by-side and histologically compared 30 days thereafter, the MSC devices showed significantly less inflammation, indicated by a 70–80% reduction in ED1 positive macrophages, and 54–56% less fibrotic vimentin immunoreactivity. Together, the data supports the use of MSC as compliant and adaptable technology for the interfacing of somatic and autonomic peripheral nerves.

The brain is an enormously complex organ structured into various regions of layered tissue. Researchers have attempted to study the brain by modeling the architecture using two dimensional (2D) in vitro cell culturing methods. While those platforms attempt to mimic the in vivo environment, they do not truly resemble the three dimensional (3D) microstructure of neuronal tissues. Development of an accurate in vitro model of the brain remains a significant obstacle to our understanding of the functioning of the brain at the tissue or organ level. To address these obstacles, we demonstrate a new method to bioprint 3D brain-like structures consisting of discrete layers of primary neural cells encapsulated in hydrogels. Brain-like structures were constructed using a bio-ink consisting of a novel peptide-modified biopolymer, gellan gum-RGD (RGD-GG), combined with primary cortical neurons. The ink was optimized for a modified reactive printing process and developed for use in traditional cell culturing facilities without the need for extensive bioprinting equipment. Furthermore the peptide modification of the gellan gum hydrogel was found to have a profound positive effect on primary cell proliferation and network formation. The neural cell viability combined with the support of neural network formation demonstrated the cell supportive nature of the matrix. The facile ability to form discrete cell-containing layers validates the application of this novel printing technique to form complex, layered and viable 3D cell structures. These brain-like structures offer the opportunity to reproduce more accurate 3D in vitro microstructures with applications ranging from cell behavior studies to improving our understanding of brain injuries and neurodegenerative diseases.

In the injured adult nervous system, re-establishment of growth-promoting molecular gradients is known to entice and guide nerve repair. However, incorporation of three-dimensional chemotactic gradients in nerve repair scaffolds, particularly in those with multi-luminal architectures, remains extremely challenging. We developed a method that establishes highly tunable three-dimensional molecular gradients in multi-luminal nerve guides by anchoring growth-factor releasing coiled polymeric fibers onto the walls of collagen-filled hydrogel microchannels. Differential pitch in the coiling of neurotrophin-eluting fibers generated sustained chemotactic gradients that appropriately induced the differentiation of Pheochromocytoma (PC12) cells into neural-like cells along an increasing concentration of nerve growth factor (NGF). Computer modeling estimated the stability of the molecular gradient within the luminal collagen, which we confirmed by observing the significant effects of neurotrophin gradients on axonal growth from dorsal root ganglia (DRG). Neurons growing in microchannels exposed to a NGF gradient showed a 60% increase in axonal length compared to those treated with a linear growth factor concentration. In addition, a two-fold increment in the linearity of axonal growth within the microchannels was observed and confirmed by a significant reduction in the turning angle ratios of individual axons. These data demonstrate the ability of growth factor-loaded polymeric coiled fibers to establish three-dimensional chemotactic gradients to promote and direct nerve regeneration in the nervous system and provides a unique platform for molecularly guided tissue repair.

Hypertension (HTN) affects over 1 billion people in the world, and while most are treated effectively with pharmacological regimens, 10–30% of them do not show a beneficial response. Electrical stimulation of the renal sympathetic, vagus and carotid sinus nerves has shown depressor effects and has been proposed as an alternative treatment for resistant hypertension (R-HTN). However, these nerves are heterogeneous in afferent/efferent composition, and their stimulation often results in unwanted side effects. We evaluated the possibility of eliciting a depressor response from stimulation of single fascicle of the somatic deep peroneal nerve (fDPN). A microchannel electrode array (µCEA) was used to stimulate the fDPN at low frequency, which induced a significant (p ≤ 0.03) transient reduction in mean arterial pressure (MAP) with no significant effects on heart rate. The depressor response was prolonged for several hours by extending the fDPN stimulation to 5 min, which induced significant reduction (17–25%) in MAP for up to 4 h. Immunofluorescence evaluation of the axonal marker, myelin, and active macrophages in the fDPN revealed no indication of nerve damage or overt inflammation in response to the procedure. This study provides evidence supporting the use of µCEA interfacing of small somatic nerve fascicles associated with cardiovascular relevant acupoints to induce significant reductions in MAP and opens the possibility of neuromodulation of small fascicles as an alternative strategy to treat R-HTN with minimal side effects. Further, the µCEA multielectrode array offers an effective tool for neuromodulation of small nerve fascicles, enabling a number of possible future medical bioelectronic applications.

Spinal cord injury (SCI) without radiographical abnormalities (SCIWORA) presents a significant challenge because of the loss of function despite an apparent normal anatomy. The cause of dysfunction is not understood, and specific treatment options are lacking. Some scoliosis corrective surgeries result in SCIWORA, where stretching of the spinal cord can lead to vascular compromise and hypoxia. The iatrogenic nature of this injury allows for the implantation of neuroprotective strategies that are designed to prevent damage. We utilized a model of atraumatic SCI to evaluate the efficacy of the sodium-channel blocker, riluzole, as a prophylactic neuroprotectant. As expected, the stretch injury caused a significant reduction in intraparenchymal oxygen in distraction (-53.09 ± 22.23%) and riluzole pre-treated distraction animals (-43.04 ± 22.86%). However, in contrast to the oxidative stress and metabolic impairments observed in vehicle-treated distraction animals, in which protein carbonylation increased significantly (5.88 ± 1.3 nmol/mL), riluzole kept these levels within the normal range (1.8 ± 1.0 nmol/mL). This neurprotection also prevented ventral motor neuron hypoplasia and pyknosis, characteristic features of this atraumatic SCI model, and maintained normal gait function (e.g., stride length and stance time). This study provides evidence for the use of prophylactic neuroprotective strategies in which thoracic or spine surgeries present the risk of causing atraumatic SCI.

Objective. Neural interfaces designed to stimulate or record electrical activity from peripheral nerves have applications ranging from the electrical modulation of nerve activity as a therapeutic option (e.g. epilepsy and untreatable depression) to the design of prosthetics. Currently, most peripheral nerve interfaces are either cuff-style devices that wrap around the target nerve or intraneural devices that are implanted within the nerve. While the latter option offers higher specificity and signal-to-noise ratio, penetrating devices can cause significant damage to the nerve due to the high degree of mechanical mismatch. Because of this, there is interest in developing penetrating devices fabricated from soft or softening materials (materials having a low elastic modulus). However, there is currently a lack of understanding regarding implantation forces required for successful insertion, which is a constraint for soft device design. Softer devices require robust designs to achieve a critical buckling force that is larger than forces experienced during device insertion. Approach. This study comprehensively assesses insertion force under different implantation conditions, with three variations for implantation speed, angle, and device tip angle, during insertion of silicon shanks in rat sciatic nerve. Additionally, we report compression modulus for rat sciatic nerve at different compression rates to inform computational modeling. Main results. We found that insertion speed and angle had significant effects on peak insertion force. We observed lower insertion forces (10-60 mN) when the device was implanted at higher angles relative to the perpendicular insertion (80-125 mN). We also demonstrate use of a nerve-stabilizing device to keep the nerve immobile during implantation. Additionally, we found that compression modulus was significantly different in small and large strain regions of the stress-strain curve with values between 1500-4500 Pa depending on compression rate. Significance. This study provides information imperative to the design and successful implementation of soft penetrating peripheral nerve interfaces.

OBJECTIVE Recent developments in peripheral nerve electrodes allow the efficient and selective neuromodulation of somatic and autonomic nerves, which has proven beneficial in specific bioelectronic medical applications. However, current most clinical devices are wired and powered by implantable batteries which suffer from several limitations. We recently developed a sub-millimeter inductively powered neural stimulator (electroparticle; EP), and in this study, we report the integration of the EP onto commercial cuff electrodes (EP-C) allowing the wireless activation of peripheral nerves. APPROACH The current output of this device was defined at different magnetic field strenghts, and with respect to external antenna distance and activation angles. In acute in vivo testing, stimulation of the rat sciatic nerve (ScN) with the EP-C was able to evoke motor responses quantified by 3D tracking of the hind limb movement. Motor recruitment curves were obtained in response to variations in magnetic field strength (0-92.91 A m-1), stimulation frequencies (2-7 Hz), and pulse widths (50-200 µs). MAIN RESULTS The results show constant output voltage throughout 50 400 stimulating cycles on a benchtop setting, and successful ScN motor activation with a 4 cm distance between external antenna and receiver. We achieved optimal motor recruitment indicated by maximizing range of hindlimb movement (6.01 ± 2.92 mm) with a magnetic field of 40.02 ± 2.85 A m-1 and 150 µs pulse width. Stimulating pulse width or frequency did not significantly influence motor recruitment. SIGNIFICANCE We confirmed that continuous stimulation for 14 min using monophasic pulses did not deleteriously affect the evoked motor responses when compared to wired charge-balanced biphasic electrical stimulation. We observed, however, a 36%-44% decrease in the evoked limb movement in both groups over time due to muscle fatigue. This study shows that the EP-C device can be used effectively for peripheral nerve neuromodulation.

Fabrication of flexible and free-standing graphene-fiber- (GF-) based microelectrode arrays with a thin platinum coating, acting as a current collector, results in a structure with low impedance, high surface area, and excellent electrochemical properties. This modification results in a strong synergistic effect between these two constituents leading to a robust and superior hybrid material with better performance than either graphene electrodes or Pt electrodes. The low impedance and porous structure of the GF results in an unrivalled charge injection capacity of 10.34 mC cm-2 with the ability to record and detect neuronal activity. Furthermore, the thin Pt layer transfers the collected signals along the microelectrode efficiently. In vivo studies show that microelectrodes implanted in the rat cerebral cortex can detect neuronal activity with remarkably high signal-to-noise ratio (SNR) of 9.2 dB in an area as small as an individual neuron.

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the 'classical' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.

Introduction Peripheral nerve injuries (PNI) are among the leading causes of physical disability in the United States. The majority of injuries occur in the upper extremities, and functional recovery is often limited. Robust animal models are critical first steps for developing effective therapies to restore function after PNI. Methods We developed an automated behavioral assay that provides quantitative measurements of volitional forelimb strength in rats. Multiple forelimb PNI models involving the median and ulnar nerves were used to assess forelimb function for up to 13 weeks post-injury. Results Despite multiple weeks of task-oriented training following injury, rats exhibit significant reductions in multiple quantitative parameters of forelimb function, including maximal pull force and speed of force generation. Discussion This study demonstrates that the isometric pull task is an effective method of evaluating forelimb function following PNI and may aid in development of therapeutic interventions to restore function. This article is protected by copyright. All rights reserved.

Multi-fingered prosthetic limbs are now capable of performing complex movements, closely simulating those of the lost human arm/hand in amputees. Conveying natural control and perception of such sophisticated robotic limbs requires direct interfacing with the nervous system of the users. Both the brain and transected peripheral nerves have been proposed as appropriate locations for such neural interfaces. In the peripheral nerves, several electrode designs have been used successfully to record motor signals, and through stimulation, used to convey sensation in long-term amputee human volunteers. Here we review the advantages and limitations of peripheral nerve interfaces (PNIs) in relation to the desired characteristics for safety and performance. We compare current PNI electrodes and materials designed to increase recording sensitivity and to achieve selective stimulation of different nerves such as those carrying mechanical sensation and limb position information. Emphasis is placed on novel electrode materials, including biological coatings, conductive polymers, and nanostructured modifications. Successful development of reliable PNIs will not only enable natural control and feel of future robotic prosthetics, but will likely be a centerpiece technology in the development of multiple bionic organs.

Large bioengineered organs such as the heart and kidney need immediate perfusion by the host vascular network to avoid ischemia, support implant survival, and prevent implant failure. Vascularization of new bioengineered tissues can be stimulated by porous scaffold design and vascular growth factors. However, comprehensive vascularization of thick tissues in vitro remains a formidable challenge. We developed a simple and reproducible vascularization method that integrates a transparent biodegradable multiluminal scaffold for guided endothelial migration stimulated by intraluminal controlled release of Vascular Endothelial Growth Factor (VEGF). Two- and three-dimensional in vitro vasculogenesis induced by human umbilical vein endothelial cells (HUVEC)/fibroblast co-cultures formed discontinuous vessel-like structures. In sharp contrast, hydrogel microchannels with luminal collagen matrix, enticed the angiogenic growth from postnatal and adult aortic explants into the agarose microchannels, forming continuous vascular tubes identified by CD31 expression, in which the number of cells can be controlled by intraluminal VEGF concentration. This study suggests that multiluminal scaffolds can be used to promote angiogenesis from mature blood vessels and form vascular networks within thick bioengineered scaffolds. This method might offer a viable alternative in the prevascularization of bio-artificial organs.

Scoliosis corrective surgery requires the application of significant multidirectional stress forces, including distraction, for correction of the curved spine deformity and the application of fixation rods. If excessive, spine distraction may result in the development of new neurological deficits, some as severe as permanent paralysis. Current animal models of spinal cord injury, however, are limited to contusion, transection, or unidirectional distraction injuries, which fail to replicate the multidirectional forces that occur during spine corrective surgery. To address such limitation, we designed a novel device that relies on intervertebral grip fixation and linear actuators to induce controllable bidirectional distraction injuries to the spine. The device was tested in three (i.e., 3, 5, and 7 mm) distention paradigms of the rat T9-T11 vertebra, and the resulting injuries were evaluated through electrophysiological, behavioral, and histological analysis. As expected, 3mm bilateral spine distractions showed no neurological deficit. In contrast, those with 5 and 7 mm showed partial and complete paralysis, respectively. The relationship between the severity of the spine distraction and injury to the spinal cord tissue was determined using glial fibrillary acidic protein immunocytochemistry for visualization of reactive astrocytes and labeling of ED1-positive activated macrophages/microglia. Our results demonstrate that this device can produce bidirectional spine distraction injuries with high precision and control and, thus, may be valuable in contributing to the testing of neuroprotective strategies aimed at preventing unintended new neurological damage during corrective spine surgery.

Introduction Women often develop pelvic floor dysfunction due to damage to the pelvic musculature during childbirth; however, the effect on pelvic floor nerves function is less understood. This study used adult rabbits to evaluate the electrophysiological and histological characteristics of the bulbospongiosus (Bsn) and pubococcygeus nerves (Pcn) in multiparity. Methods Compound nerve action potentials (CNAP) were compared between age‐matched nulliparous and multiparous animals and associated to the histological characteristics of myelinated axons from the Bsn and Pcn nerves. The extensor digitorum longus nerve (EDLn) was used as negative control. Data were analyzed with unpaired two‐tailed Student's t test or Mann–Whitney U test to determine significant differences between groups. Results The onset and peak latencies, duration, and conduction velocity of the motor fibers in these pelvic nerves were not significantly different between nulliparous and multiparous animals. However, the peak‐to‐peak amplitude and area of the CNAP in both Bsn and Pcn were reduced in multiparous rabbits. Histology showed a higher percentage of axons with myelin disorganization caused by multiparity in these pelvic nerves. Together, the data indicate a reduction in the number of functional pelvic axons due to multiparity. As expected, no effect of parity was observed in the EDLn controls. Conclusions Present findings demonstrated that multiparity affects myelination and consequently conduction properties in the small pelvic floor nerves.

A biocompatible neural microprobe constructed using well-established SU-8 microfabrication techniques is described that was designed to record fiber spike signals from regenerated axons within peripheral nerves. These microprobes features bipolar longitudinal gold electrodes recessed below the surface within ldquogroovesrdquo designed to guide the growth of regenerating axons along the length of the grooves and limit the number of fibers that come in contact with the longitudinal electrodes. In addition, screening microprobe toxicity using cultures of human skin fibroblasts, the biocompatibility of these SU-8 microprobes for neural interface applications, in particular, was specifically verified using primary cultures of two sensitive cell types found in peripheral nerves: purified Schwann cells and explanted dorsal root ganglion (DRG) neurons and their fibers. The SU-8 microprobes were surgically implanted into transected rat Sciatic nerves within a unique peripheral nerve regeneration tube. Long-term fiber spike signals were recorded with these SU-8 microprobes in 13 chronically implanted rats for periods from 4 to 51 weeks without any signs of tissue damage or inflammatory reaction.