Accelerate Nerve Regeneration Research Articles

The effect of subthreshold continuous electrical stimulation on the facial function of patients with Bell’s palsy

Conclusion: The drug regimen plus electrical stimulation was more effective in treating Bell’s palsy than the conventional drug treatment alone. The effectiveness of such a sub-threshold, continuous, low frequency electrical stimulation suggests a new therapeutic approach to accelerate nerve regeneration and improve functional recovery after injury. Objectives: The purpose of this study was to determine whether sub-threshold, continuous electrical stimulation at 20 Hz facilitates functional recovery of patients with Bell’s palsy. Materials and methods: The authors performed a prospective randomized study that included 60 patients with mild-to-moderate grade Bell’s palsy (HB grade ≤4, SB grade ≥40), to evaluate the effect of developed electrical stimulation on the resolution of symptoms. Thirty patients were treated with prednisolone or/and acyclovir plus electrical stimulation within 7 days of the onset of symptoms. The other 30 patients were treated with only prednisolone or/and acyclovir as a control group. Results: The overall rate of patient recovery among those treated with prednisolone or/and acyclovir plus electrical stimulation (96%) was significantly better (p < 0.05) than the rate among those treated with only prednisolone or/and acyclovir (88%).

Acceleration of Schwann Cell Growth Co-cultured with Amniotic Membrane and Amniotic Fluid Stem Cells: N/A - Not a clinical study

Acceleration of Schwann Cell Growth Co-cultured with Amniotic Membrane and Amniotic Fluid Stem Cells: N/A - Not a clinical study

Effects of medium flow on axon growth with or without nerve growth factor

Axon growth is a crucial process in regeneration of damaged nerves. On the other hand, elongation of nerve fibers in the epidermis has been observed in skin of atopic dermatitis patients. Thus, regulation of nerve fiber extension might be an effective strategy to accelerate nerve regeneration and/or to reduce itching in pruritus dermatosis. We previously demonstrated that neurons and epidermal keratinocytes similarly contain multiple receptors that are activated by various environmental factors, and in particular, keratinocytes are influenced by shear stress. Thus, in the present study, we evaluated the effects of micro-flow of the medium on axon growth in the presence or absence of nerve growth factor (NGF), using cultured dorsal-root-ganglion (DRG) cells. The apparatus, AXIS™, consists of two chambers connected by a set of microgrooves, through which signaling molecules and axons, but not living cells, can pass. When DRG cells were present in chamber 1, NGF was present in chamber 2, and micro-flow was directed from chamber 1 to chamber 2, axon growth was significantly increased compared with other conditions. Acceleration of axon growth in the direction of the micro-flow was also observed in the absence of NGF. These results suggest that local micro-flow might significantly influence axon growth.

Effect of nano-hydroxyapatite-coated magnetic nanoparticles on axonal guidance growth of rat dorsal root ganglion neurons.

Proper extracellular substrate can stimulate neural regeneration in nerve tissue engineering, including magnetic nanoparticles (iron oxide nanoparticles, Fe3 O4 ), but they are always neurotoxic, with low saturation magnetization and so on. These nanomaterials cannot be used to stimulate the growth and elongation of axons. Therefore, this work attempts to overcome these deficiencies. Nano-hydroxyapatite (n-HA) coated magnetic nanoparticles were using an ultrasound-assisted co-precipitation method. X-ray diffraction and transmission electron microscopy were used to characterize the structure and chemical composition of the produced samples. These synthesized nanomaterials were added into the primary cultured dorsal root ganglion (DRG) neurons; our results showed that n-HA-coated magnetic nanoparticles (Fe3 O4 +n-HA) can effectively increase cell viability and promote axonal elongation, which enhanced saturation magnetization. In addition, we demonstrated that axonal guidance cues Netrin-1 increase significantly after n-HA-coated magnetic nanoparticles treatment by Western blots assay. n-HA-coated magnetic particles maybe applied to enhance or accelerate nerve regeneration, and it may provide guidance for regenerating axons in future.

Update in facial nerve paralysis

To present the recent advances in the treatment of facial paralysis, emphasizing the emerging technologies. This review will summarize the current state of the art in the management of facial paralysis and discuss the advances in nerve regeneration, facial reanimation, and use of novel biomaterials. This review includes surgical innovations in reinnervation and reanimation as well as progress with bioelectrical interfaces. The last decade has witnessed major advances in the understanding of nerve injury and approaches for management. Key innovations include strategies to accelerate nerve regeneration, provide tissue-engineered constructs that may replace nonfunctional nerves, approaches to influence axonal guidance, limiting of donor-site morbidity, and optimization of functional outcomes. Approaches to muscle transfer continue to evolve, and new technologies allow for electrical nerve stimulation and use of artificial tissues. The fields of biomedical engineering and facial reanimation increasingly intersect, with innovative surgical approaches complementing a growing array of tissue engineering tools. The goal of treatment remains the predictable restoration of natural facial movement, with acceptable morbidity and long-term stability. Advances in bioelectrical interfaces and nanotechnology hold promise for widening the window for successful treatment intervention and for restoring both lost neural inputs and muscle function.

Peripheral nerve regeneration through a silicone chamber implanted with negative carbon ions: Possibility to clinical application

Abstract We investigated whether a tube with its inner surface implanted with negative-charged carbon ions (C − ions) would enable axons to extend over a distance greater than 10 mm. The tube was found to support nerves regenerating across a 15-mm-long inter-stump gap. We also investigated whether a C − ion-implanted tube pretreated with basic fibroblast growth factor (bFGF) promotes peripheral nerve regeneration. The C − ion implanted tube accelerated nerve regeneration, and this effect was enhanced by bFGF. Silicone treated with C − ions showed increased hydrophilic properties and cellular affinity, and axon regeneration was promoted with this increased biocompatibility.

In vitro and in vivo optimization of infrared laser treatment for injured peripheral nerves

Repair of peripheral nerve injuries remains a major challenge in restorative medicine. Effective therapies that can be used in conjunction with surgical nerve repair to improve nerve regeneration and functional recovery are being actively investigated. It has been demonstrated by a number of peer reviewed publications that photobiomodulation (PBM) supports nerve regeneration, reinnervation of the denervated muscle, and functional recovery after peripheral nerve injury. However, a key issue in the use of PBM as a treatment for peripheral nerve injury is the lack of parameter optimization for any given wavelength. The objective of this study was to demonstrate that for a selected wavelength effective in vitro dosing parameters could be translated to effective in vivo parameters. Comparison of infra-red (810 and 980 nm wavelengths) laser treatment parameters for injured peripheral nerves was done beginning with a series of in vitro experiments using primary human fibroblasts and primary rat cortical neurons. The primary rat cortical neurons were used for further optimization of energy density for 980 nm wavelength light using measurement of total neurite length as the bioassay. For these experiments, the parameters included a 1 W output power, power density of 10 mW/cm(2) , and energy densities of 0.01, 0.1, 0.5, 2, 10, 50, 200, 1,000, and 5,000 mJ/cm(2) . For translation of the in vitro data for use in vivo it was necessary to determine the transcutaneous penetration of 980 nm wavelength light to the level of the peroneal nerve. Two anesthetized, male White New Zealand rabbits were used for these experiments. The output power of the laser was set at 1.0 or 4.0 W. Power density measurements were taken at the surface of the skin, sub-dermally, and at the level of the nerve. Laser parameters used in the in vivo studies were calculated based on data from the in vitro studies and the light penetration measurements. For the in vivo experiments, a total of 22 White New Zealand rabbits (2.34-2.89 kg) were used. Translated dosing parameters were refined in a pilot study using a transection model of the peroneal nerve in rabbits. Output powers of 2 and 4 W were tested. For the final set of in vivo experiments, the same transection nerve injury model was used. An energy density of 10 mW/cm(2) at the level of the peroneal nerve was selected and the laser parameters were further refined. The dosing parameters used were: 1.5 W output power, 43 seconds exposure, 8 cm(2) area and a total energy of 65 J. In vitro, 980 nm wavelength light at 10 mW/cm(2) significantly improved neurite elongation at energy densities between 2 and 200 mJ/cm(2) . In vivo penetration of the infrared light measured in anesthetized rabbits showed that on average, 2.45% of the light applied to the skin reached the depth of the peroneal nerve. The in vivo pilot study data revealed that the 4 W parameters inhibited nerve regeneration while the 2 W parameters significantly improved axonal regrowth. For the final set of experiments, the irradiated group performed significantly better in the toe spread reflex test compared to the control group from week 7 post-injury, and the average length of motor endplates returned to uninjured levels. The results of this study demonstrate that treatment parameters can be determined initially using in vitro models and then translated to in vivo research and clinical practice. Furthermore, this study establishes that infrared light with optimized parameters promotes accelerated nerve regeneration and improved functional recovery in a surgically repaired peripheral nerve.

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3V, 20Hz, 20min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro-Gold retrograde-labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain-derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF-mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.

Acceleration of Peripheral Nerve Regeneration through Asymmetrically Porous Nerve Guide Conduit Applied with Biological/Physical Stimulation

Sufficient functional restoration of damaged peripheral nerves is a big clinical challenge. In this study, a nerve guide conduit (NGC) with selective permeability was prepared by rolling an asymmetrically porous polycaprolactone/Pluronic F127 membrane fabricated using a novel immersion precipitation method. Dual stimulation (nerve growth factor [NGF] as a biological stimulus and low-intensity pulse ultrasound [US] as a physical stimulus) was adapted to enhance nerve regeneration through an NGC. The animal study revealed that each stimulation (NGF or US) has a positive effect to promote the peripheral nerve regeneration through the NGC, however, the US-stimulated NGC group allowed more accelerated nerve regeneration compared with the NGF-stimulated group. The NGC group that received dual stimulation (NGF and US) showed more effective nerve regeneration behavior than the groups that received a single stimulation (NGF or US). The asymmetrically porous NGC with dual NGF and US stimulation may be a promising strategy for the clinical treatment of delayed and insufficient functional recovery of a peripheral nerve.

Incorporation of parallel electrospun fibers for improved topographical guidance in 3D nerve guides

Three dimensional (3D) conduits facilitate nerve regeneration. Parallel microfibers have been shown to guide axon extension and Schwann cell migration on flat sheets via topographical cues. However, incorporation of aligned microfibers into 3D conduits to accelerate nerve regeneration has proven challenging. We report an electrospinning technique to incorporate parallel microfibers into 3D constructs at high surface areas while retaining an open architecture. The nerve guide consists of many microchannels lined with a thin layer of longitudinally-aligned microfibers. This design aims to maximize benefits of topographical cues without inhibiting cellular infiltration. We support this hypothesis by demonstrating efficient cell infiltration in vitro. Additionally, this new technique reduces wall thickness compared to our previous design, providing a greater total area for tissue growth. This approach results in an architecture that very closely mimics the structure of decellularized nerve but with larger microchannel diameters to encourage cell infiltration. We believe that reproducing the native architecture is the first step toward matching autograph efficacy. Furthermore, this design can be combined with other biochemical cues to promote nerve regeneration.

Light‐emitting diode therapy induces analgesia and decreases spinal cord and sciatic nerve tumour necrosis factor‐α levels after sciatic nerve crush in mice

Neuropathic pain is severely debilitating and resistant to pharmacological approaches; therefore, the study of therapies to complement its treatment is especially relevant. In a case report study, light-emitting diode therapy (LEDT) has shown analgesic activity as well as reduced the expression of pro-inflammatory cytokines in a rabbit osteoarthritis model and in calcaneal tendinitis in rats. Although LEDT stimulated morphofunctional recovery after nerve injury in rats, its effect against neuropathic pain has not been tested. To that purpose, mice under anaesthesia were subjected to the sciatic nerve crush (SNC) model. On the seventh post-operative day, after determining analgesic dose (energy density in joules), LEDT (950 nm, 80 mW/cm2, 2.5 J/cm2 ) was irradiated, daily for a period of 15 days, on the skin over the crush site. Compared with the SNC group, LEDT reduced mechanical hypersensitivity but not cold hypersensitivity which is induced by SNC, decreased spinal cord and sciatic nerve levels of tumour necrosis factor alpha (TNF-α) but did not alter interleukin (IL)-1β and IL-10 levels, and finally, failed to accelerate motor functional recovery and morphological nerve regeneration. Taken together, these data provide first-hand evidence of LEDT effectiveness against neuropathic pain induced by SNC, with corresponding decrease of pro-inflammatory cytokine levels, both in the sciatic nerve and in the spinal cord, although at a small analgesic dose, LEDT failed to accelerate nerve regeneration.

(−)-Epigallocatechin-3-gallate (EGCG) attenuates peripheral nerve degeneration in rat sciatic nerve crush injury

Recently, we have shown that green tea (GT) consumption improves both reflexes and sensation in unilateral chronic constriction injury to the sciatic nerve. Considering the substantial neuroprotective properties of GT polyphenols, we sought to investigate whether (−)-epigallocatechin-3-gallate (EGCG) could protect the sciatic nerve and improve functional impairments induced by a crushing injury. We also examined whether neuronal cell apoptosis induced by the crushing injury is affected by EGCG treatment.Histological examination of sciatic nerves from EGCG-treated (50mg/kg; i.p.) showed that axonotmized rats had a remarkable axonal and myelin regeneration with significant decrease in the number of myelinated axonal fibers compared to vehicle-treated crush group. Similarly, ultrastructural evaluation of EGCG-treated nerves displayed normal unmyelinated and myelinated axons with regular myelin sheath thickness and normalized appearance of Schmidt–Lantermann clefts. Extracellular matrix displayed normal collagen fibers appearance with distinctively organized distribution similar to sham animals. Analysis of foot position and extensor postural thrust test showed a progressive and faster recovery in the EGCG-treated group compared to vehicle-treated animals. EGCG-treated rats showed significant increase in paw withdrawal thresholds to mechanical stimulation compared to vehicle-treated crush group. EGCG treatment also restored the mRNA expression of Bax, Bcl-2 and survivin but not that of p53 to sham levels on days 3 and 7 post-injury.Our results demonstrate that EGCG treatment enhanced functional recovery, advanced morphological nerve rescue and accelerated nerve regeneration following crush injury partly due to the down regulation of apoptosis related genes.

Blockade of IL-6 receptor accelerates nerve regeneration in experimental sciatic nerve crush injury

IL-6 is known as a multifunctional cytokine regulating immune response and inflammation. Many studies have shown that IL-6 affects the nervous system. However, the involvement of IL-6 in the nervous system, particularly in the peripheral nervous system (PNS), remains incompletely understood. Here, we assessed the effect of rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) in the treatment of experimental sciatic nerve crush injury in mice. The mice were given a single intraperitoneal injection (2 mg/head) of MR16-1 or Rat IgG as a control antibody the day before nerve crush injury. Treatment with MR16-1 significantly promoted sciatic functional recovery, and histological remyelination was greater than that by Rat IgG treatment. Furthermore, IL-6 expression was confirmed in the nerve after nerve crush injury in the Rat IgG group, but not in the MR16-1 group. MR16-1 also decreased the number of infiltrating macrophages. Additionally, IL-6 decreased the expression of nerve growth factor (NGF) mRNA in cultured primary mouse Schwann cells, and this decrease in NGF expression by IL-6 was reversed with MR16-1. These findings suggest that MR16-1 may inhibit the infiltration of macrophages and the decrease of NGF in Schwann cells, resulting in the acceleration of nerve regeneration. Rec.10/30/2012, Acc.1/24/2013, pp156-163

Low-Level Laser-Accelerated Peripheral Nerve Regeneration within a Reinforced Nerve Conduit across a Large Gap of the Transected Sciatic Nerve in Rats

This study proposed a novel combination of neural regeneration techniques for the repair of damaged peripheral nerves. A biodegradable nerve conduit containing genipin-cross-linked gelatin was annexed using beta-tricalcium phosphate (TCP) ceramic particles (genipin-gelatin-TCP, GGT) to bridge the transection of a 15 mm sciatic nerve in rats. Two trigger points were irradiated transcutaneously using 660 nm of gallium-aluminum arsenide phosphide (GaAlAsP) via laser diodes for 2 min daily over 10 consecutive days. Walking track analysis showed a significant improvement in sciatic functional index (SFI) (P < 0.01) and pronounced improvement in the toe spreading ability of rats undergoing laser stimulation. Electrophysiological measurements (peak amplitude and area) illustrated by compound muscle action potential (CMAP) curves demonstrated that laser stimulation significantly improved nerve function and reduced muscular atrophy. Histomorphometric assessments revealed that laser stimulation accelerated nerve regeneration over a larger area of neural tissue, resulting in axons of greater diameter and myelin sheaths of greater thickness than that observed in rats treated with nerve conduits alone. Motor function, electrophysiological reactions, muscular reinnervation, and histomorphometric assessments all demonstrate that the proposed therapy accelerated the repair of transected peripheral nerves bridged using a GGT nerve conduit.

Accelerated nerve regeneration using induced pluripotent stem cells in chitin–chitosan–gelatin scaffolds with inverted colloidal crystal geometry

Polymeric biomaterials with regular pore structure can generate distinctive properties in various biomedical applications. This study presents scaffolds comprising chitin, chitosan, and gelatin with pore geometry of inverted colloidal crystals (ICC) for guiding differentiation of induced pluripotent stem (iPS) cells toward neurons. Chitin–chitosan–gelatin ICC scaffolds were fabricated by self-assembly, crosslinking, infiltration, dehydration, and particle leaching. The results revealed that ethanol as a dispersion medium yielded a higher regularity of colloidal template than ethylene glycol. For the adhesion of iPS cells, freeform constructs were more efficient than ICC constructs. The reverse was true for the viability of iPS cells. The quantity of stage-specific embryonic surface antigen-1 in cultured freeform construct was larger than that in cultured ICC construct, indicating that the former preserved more phenotypic characteristics of iPS cells than the latter. Moreover, β III tubulin-identified region in ICC construct was larger than that in freeform construct, demonstrating that the differentiation of iPS cells toward neurons in ICC construct was faster than that in freeform construct. ICC topography in chitin–chitosan–gelatin scaffolds can accelerate neuronal differentiation of iPS cells.

Geldanamycin accelerated peripheral nerve regeneration in comparison to FK-506 in vivo

FK-506 accelerates nerve regeneration and improves functional recovery in vivo; its immunosuppressive properties, however, limit its clinical utility. Geldanamycin (GA), a non-immunosuppressive agent, shares a common binding target (heat shock protein 90) with FK-506 and may accelerate nerve regeneration through a similar mechanism. GA has been shown to augment neurite outgrowth in vitro but has not been tested in vivo. The current study investigated the effect of GA on the rate of axonal regeneration and functional recovery following peripheral nerve injury. In the first experiment, Thy1-GFP transgenic rats underwent serial transmuscular imaging to quantify the rate of axonal regeneration following saphenous nerve crush injury. In subsequent experiments, Lewis rats underwent tibial nerve crush or transection-and-repair injuries and were assessed for functional recovery by walking track analysis. All animals were randomized to receive daily administration of FK-506 (2mg/kg), GA (0.2mg/kg), or a control vehicle (dimethyl sulfoxide, 1mL/kg) starting 3days prior to injury. Both GA and FK-506 significantly increased the rate of axonal regeneration following crush injury in Thy1-GFP rats. In Lewis rats undergoing tibial nerve crush injury, earlier functional recovery occurred at day 5 and day 6 in animals treated with FK-506 and GA respectively, vs. day 13 for controls. Over a truncated 21-day timeframe, Lewis rats undergoing tibial nerve transection-and-repair injury and treated with FK-506 regained function at day 16, whereas those treated with GA or the control vehicle did not regain normal function. GA-treated animals, however, did exhibit significant functional improvement vs. controls. The current study demonstrated that GA accelerates axonal regeneration and enhances functional recovery in vivo. Its ability to increase the rate at which peripheral axons regenerate is comparable to that of FK-506. GA, however, did not match the performance of FK-506 in injury models where Wallerian degeneration (WD) is ongoing in the distal stump. This provides evidence that FK-506 accelerates axonal regeneration through two parallel mechanisms: the first being its well-established effect on neurons; the second is likely a newly described, as-yet poorly defined mechanism that affects WD. Finally, given the decrease in observed toxicity with GA administration, it might be a suitable non-immunosuppressive alternative to FK-506 for accelerating peripheral nerve regeneration in cases of clinical nerve injury.

Electrical Stimulation to Conductive Scaffold Promotes Axonal Regeneration and Remyelination in a Rat Model of Large Nerve Defect

BackgroundElectrical stimulation (ES) has been shown to promote nerve regeneration when it was applied to the proximal nerve stump. However, the possible beneficial effect of establishing a local electrical environment between a large nerve defect on nerve regeneration has not been reported in previous studies. The present study attempted to establish a local electrical environment between a large nerve defect, and examined its effect on nerve regeneration and functional recovery.Methodology/FindingsIn the present study, a conductive scaffold was constructed and used to bridge a 15 mm sciatic nerve defect in rats, and intermittent ES (3 V, 20 Hz) was applied to the conductive scaffold to establish an electrical environment at the site of nerve defect. Nerve regeneration and functional recovery were examined after nerve injury repair and ES. We found that axonal regeneration and remyelination of the regenerated axons were significantly enhanced by ES which was applied to conductive scaffold. In addition, both motor and sensory functional recovery was significantly improved and muscle atrophy was partially reversed by ES localized at the conductive scaffold. Further investigations showed that the expression of S-100, BDNF (brain-derived neurotrophic factor), P0 and Par-3 was significantly up-regulated by ES at the conductive scaffold.Conclusions/SignificanceEstablishing an electrical environment with ES localized at the conductive scaffold is capable of accelerating nerve regeneration and promoting functional recovery in a 15 mm nerve defect in rats. The findings provide new directions for exploring regenerative approaches to achieve better functional recovery in the treatment of large nerve defect.

Efficacy of Short-Term FK506 Administration on Accelerating Nerve Regeneration

Background. The slow rate of nerve regeneration following injury can cause extended muscle denervation, leading to irreversible muscle atrophy, fibrosis, and destruction of motor endplates. The immunosuppressant FK506 (tacrolimus) has been shown to accelerate the rate of nerve regeneration and functional recovery. However, the toxic and immunosuppressive properties of FK506 make it undesirable for long-term use. Objective. To take advantage of the regeneration-enhancing effects of FK506 but avoid the potential adverse effects of long-term administration, the current study evaluates and quantifies the efficacy of short-term FK506 treatment in rat models. Methods. Clinically relevant transection and graft models were evaluated, and walking track analysis (WTA) was used to evaluate functional recovery. FK506 was administered for 5 and 10 days post transection injury and 10 and 20 days post graft injury. Both groups involving a short course were compared with the continuous administration group. Results. In the transection model, FK506 was administered for 5 and 10 days postoperatively. WTA demonstrated that 10 days of FK506 administration was sufficient to reduce functional recovery time by 29% compared with negative controls. In the graft model, FK506 was administered for 10 and 20 days postoperatively. Short treatment courses of 10 and 20 days reduced recovery time by 15% and 21%, respectively, compared with negative controls. Analysis of blood–nerve barrier (BNB) integrity demonstrated that FK506 facilitated early reconstitution of the BNB. Conclusions. The results of this study indicate that short-term FK506 delivery following nerve injury imparts a significant therapeutic effect.

Poly-l-lysine-coated magnetic nanoparticles as intracellular actuators for neural guidance

PurposeIt has been proposed in the literature that Fe3O4 magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons. MNPs could create mechanical tension that stimulates the growth and elongation of axons. Particles suitable for this purpose should possess (1) high saturation magnetization, (2) a negligible cytotoxic profile, and (3) a high capacity to magnetize mammalian cells. Unfortunately, the materials currently available on the market do not satisfy these criteria; therefore, this work attempts to overcome these deficiencies.MethodsMagnetite particles were synthesized by an oxidative hydrolysis method and characterized based on their external morphology and size distribution (high-resolution transmission electron microscopy [HR-TEM]) as well as their colloidal (Z potential) and magnetic properties (Superconducting QUantum Interference Devices [SQUID]). Cell viability was assessed via Trypan blue dye exclusion assay, cell doubling time, and MTT cell proliferation assay and reactive oxygen species production. Particle uptake was monitored via Prussian blue staining, intracellular iron content quantification via a ferrozine-based assay, and direct visualization by dual-beam (focused ion beam/scanning electron microscopy [FIB/SEM]) analysis. Experiments were performed on human neuroblastoma SH-SY5Y cell line and primary Schwann cell cultures of the peripheral nervous system.ResultsThis paper reports on the synthesis and characterization of polymer-coated magnetic Fe3O4 nanoparticles with an average diameter of 73 ± 6 nm that are designed as magnetic actuators for neural guidance. The cells were able to incorporate quantities of iron up to 2 pg/cell. The intracellular distribution of MNPs obtained by optical and electronic microscopy showed large structures of MNPs crossing the cell membrane into the cytoplasm, thus rendering them suitable for magnetic manipulation by external magnetic fields. Specifically, migration experiments under external magnetic fields confirmed that these MNPs can effectively actuate the cells, thus inducing measurable migration towards predefined directions more effectively than commercial nanoparticles (fluidMAG-ARA supplied by Chemicell). There were no observable toxic effects from MNPs on cell viability for working concentrations of 10 μg/mL (EC25 of 20.8 μg/mL, compared to 12 μg/mL in fluidMAG-ARA). Cell proliferation assays performed with primary cell cultures of the peripheral nervous system confirmed moderate cytotoxicity (EC25 of 10.35 μg/mL).ConclusionThese results indicate that loading neural cells with the proposed MNPs is likely to be an effective strategy for promoting non-invasive neural regeneration through cell magnetic actuation.

Direct Current Electrical Stimulation of Acupuncture Needles for Peripheral Nerve Regeneration: An Exploratory Case Series

To examine the therapeutic effect of a novel therapeutic method based on electroacupuncture with intermittent direct current (DCEA) and associated adverse events in patients with peripheral nerve damage and a poor clinical prognosis. In seven older patients with peripheral nerve damage (neurapraxia 2, axonotmesis 4, neuromesis 1), an acupuncture needle connected to an anode electrode was inserted proximal to the site of the injury along the route of the nerve, while the cathode electrode was inserted into the innervated muscle, and DCEA was performed (100 Hz for 20 min, weekly). Muscular paralysis was evaluated weekly with manual muscle testing, the active range of motion of joints related to the muscular paralysis and, when necessary, needle electromyography. Adverse events were also recorded during the course of the treatment. Complete functional recovery was observed in the two cases with neurapraxia and two with axonotmesis, while one axonotmesis case achieved improvement and the other showed reinnervation potential without functional recovery. No improvement was observed in the neurotmesis case. Pigmentation of the skin where the anode needle was inserted occurred in three cases. Although there was no definite causal link, one case showed excessive formation and resorption of bone in the area close to the cathode needle site. Accelerated nerve regeneration caused by DCEA may contribute to recovery. The skin pigmentation and callus formation suggest that the shape of the anode electrode, current intensity and other factors should be examined to establish a safer treatment method.