Establishment Of Two Modalities Of Electrical Stimulation For Promoting Nerve Regeneration And Their Mechanisms

Lengthy peripheral nerve defect has been posing a clinical challenge fororthopedic surgeons over the past decades. Tissue engineering nerve scaffoldshave been considered as promising alternatives to nerve autograft in bridginglengthy nerve defects. Nerve scaffolds provide bridges through which injuredaxons regenerate into the distal nerve stump to restore motor function. However,due to the limited regenerative capacity of injured axons, a long period of timewas generally required for regenerating axons to cross the graft, whichsignificantly limits the outcome of nerve injury repair. Therefore, accelerating therate of axonal regeneration and shortening the time required for axons to cross thegraft may help to improve motor functional recovery in the treatment of largenerve defects. It is well known that the peripheral nervous system is in acomplicated electrical environment. The electrical properties of neurons changeduring the development and reconstruction of nervous system. It has been shownthat electrical stimulation (ES) is capable of activating the regenerative programof injured nerve, guiding the linear growth of nerve growth cone, and thuspromoting nerve regeneration. Therefore, ES is one of the effective ways topromote nerve regeneration. However, studies concerning the effect of ES onnerve regeneration have been performed only in animal models of crush injury, transected injury or a short nerve gap. The effect of ES on nerve regeneration andmotor functional recovery over a lengthy nerve defect has been rare. Therefore,establishment of ES modalities which can accelerate nerve regeneration holdsgreat potential to improve the outcome of lengthy nerve defects. In the currentstudy, two modalities of ES were successfully established, which were capable ofpromoting functional recovery when they were applied together with nervescaffold with longitudinally oriented micro-channels. The two modalities of ESwere listed as follows: (1) Single short time ES to proximal nerve stump canactivate damaged neurons, accelerate nerve regeneration, shorten the timerequired for regenerating fibers to cross scaffold, and promote functionalrecovery when nerve defect was bridged with scaffold with longitudinallyoriented micro-channels; (2) Intermittent ES which was localized to conductivescaffold can restore an electrical environment at the site of nerve defect, and isbeneficial for nerve injury repair. Further studies were performed to identify theeffect of ES on the biological behaviors of Schwann cells. We found that properES is capable of regulating the biological behaviors of Schwann cells, includingproliferation, cell adhesion, spreading, as well as the expression and secretion ofNGF and BDNF. The regulatory effect of ES on Schwann cell might partiallycontribute to the beneficial effect of ES on nerve regeneration. The current studyenriches the electrochemical mechanism for nerve regeneration, and providestheoretical supports for the application of ES in the treatment of large nervedefects. The whole studies were divided into three parts:Partâ… : Electrical stimulation of the proximal nerve stump acceleratesaxonal regeneration and functional recovery through scaffolds withlongitudinally oriented micro-channelsBackgrounds. Tissue engineering nerve scaffold provides a bridge throughwhich regenerating nerve fibers enter the distal nerve stump. However, due to thelimited regenerative capacity of injured axons, a long period of time was generally required for regenerating axons to cross the graft, which significantlylimits the outcome of nerve injury repair. Therefore, accelerating the rate ofaxonal regeneration and shortening the time required for axons to cross the graftmay help to improve the motor functional recovery in the treatment of large nervedefects. ES to the proximal nerve stump has been found to enhance theregenerative capacity of axotomized motor and sensory neurons. Therefore, thecombined application of ES and tissue engineering graft holds great potential inimproving the outcome of large nerve defect.Ojective. To determine whether ES to the proximal nerve stump can accelerateaxonal regeneration and functional recovery in a 15-mm sciatic nerve defect inrats, which was bridged with our newly developed scaffold with longitudinallyoriented micro-channels.Methods. A 15-mm excision of the sciatic nerve was bridged with a chitosanscaffold with longitudinally oriented micro-channels. In half the animals withchitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at20 Hz immediately after the nerve repair with scaffolds. Axonal regeneration wasinvestigated by retrograde labeling and morphometric analysis. The rate of motorfunctional recovery was evaluated by electrophysiological studies, behavioraltests of stepping, and histological appearance of the target muscles.Results. Axonal regeneration and motor functional recovery were improved byES in animals that received longitudinal pore grafts as compared with others. Themaximal number of axons that regenerated across the longitudinal graft wasachieved 2 to 4 weeks earlier in rats with ES. In addition, the latency ofcompound muscle action potentials (CMAPs), the peak amplitude of CMAPs,and nerve conduction velocity (NCV) were improved by ES. Stepping indiceswere better, with less atrophy of target muscle in ES rats managed with longitudinal pores.Conclusion. The scaffold used in this study has longitudinally orientedmicro-channels, which have been shown to be able to guide the linear growth ofregenerating fibers. In addition, brief ES to the proximal nerve stump acceleratesaxonal regeneration and shortens the time required for regenerating axons tocross the scaffold, which leads to improved functional recovery after lengthynerve defect.Partâ…¡: ES promotes axonal regeneration and remyelination throughconductive scaffold with longitudinally oriented micro-channelsBackgrounds. The peripheral nervous system is in a complicated electricalenvironment. The electrical properties of neurons change during the developmentand reconstruction of nervous system. In case of peripheral nerve defect, theelectrical environment at the local site of nerve defect is missing, whichinevitably influence the outcome of peripheral nerve injury repair. Therefore,restoration of electrical environment at the site of nerve defect holds greatpotential in improving the outcome of nerve gap repair.Objective. To investigate the possibility of joint application of ES and conductivescaffold in restoration of electrical environment at the site of nerve defect, andexamine the efficacy of such a strategy in bridging a 15mm nerve defect in rats.Methods. Conductive scaffolds with longitudinally oriented micro-channels werefabricated with conductive PPy/chitosan composite. A 15 mm sciatic nerve defectwas made in Sprague-Dawley rats (n=120). The nerve defects were bridged eitherwith conductive PPy/chitosan scaffolds (n=60) or with non-conductive chitosanscaffolds (n=60). One hour ES (3V, 20Hz) was applied every two days for 8 timesto half of the rats which were bridged with conductive scaffold (CS+ES group)and non-conductive scaffold (NCS+ES group). The remaining rats which were bridged with conductive scaffold (CS-ES group) and non-conductive scaffold(NCS-ES group) without ES were served as controls. The effect of jointapplication of ES and conductive scaffold on axonal regeneration was examinedusing morphometric analysis and retrograde labeling. Their effect on functionalrecovery was investigated by electrophysiological study, behavioral study andhistological appearance of target muscle. The protein profile of S-100, BDNF, P0and Par-3 at the site of conductive scaffold at 3 weeks after surgery wasexamined by Western blot.Results: We found that axon regeneration parameters, including the total area ofregenerated axons, the number of myelinated axons, the mean diameter andmyelination degree of the regenerated axons, as well as the number of fluoro-goldretrograde labeled motoneurons and sensory neurons, were significantlyimproved or enhanced in the CS+ES group compared to that in the CS-ES,NCS+ES and NCS-ES groups. The functional recovery and muscle atrophy werealso significantly improved in the CS+ES group. In addition, the expression ofS-100, BNDF, P0 and Par-3 was significantly unregulated by ES applied toconductive scaffolds.Conclusion: Joint application of conductive scaffold and ES is capable ofpromoting nerve regeneration and functional recovery in a 15 mm nerve defect inrats. The efficacy of such a strategy in a larger nerve defect in larger animalspecies still needs to be identified in future studies.Partâ…¢: The mechanism underlying the beneficial effect of ES on nerveregeneration: Electrical regulation of the biological behaviors of SchwanncellsBackgrounds. ES has been shown to be capable of dramatically enhancingneurite outgrowth and accelerating peripheral nerve regeneration in animal models of nerve injury. The mechanism underlying the beneficial effect of ES onnerve regeneration is still unclear. Most of previous work focused on the effect ofES on axonal elongation and neuron cell bodies. The possible effect of ES onSchwann cells (the glial cell in the peripheral nervous system) is ignored, whichmight be responsible, at least in part, for the beneficial effect of ES on nerveregeneration.Objective. To examine the putative regulatory effect of ES on the biologicalbehaviors of Schwann cells, and examine the involvement of calcium ions in thisprocess.Methods. A biodegradable conductive composite made of conductivepolypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared inorder to electrically stimulate Schwann cells. The tolerance of Schwann cells toES was examined by a cell apoptosis assay. The growth of Schwann cells wascharacterized using DAPI staining and a MTT assay. The mRNA and proteinlevels of NGF and BDNF in Schwann cells were assayed by RT-PCR andWestern blotting, and the amount of NGF and BDNF secreted was determined byan ELISA assay. In addition, the possible involvement of calcium ions wasexamined using a calcium imaging technique, and the pathways through whichcalcium ion plays its role were characterized by pharmacological interventions.Results. The PPy/chitosan membranes supported cell adhesion, spreading, andproliferation with or without ES. High intensity of ES (600mV/mm and 1000 mV/mm) resulted in increased apoptosis in Schwann cells, while ES at 100 mV/mmwas able to promote the proliferation and adhesion of Schwann cells.Interestingly, ES applied through the PPy/chitosan composite dramaticallyenhanced the expression and secretion of NGF and BDNF as compared to controlcells without ES. Calcium imaging showed that ES-induced NGF release required calcium influx through T-type voltage-gated calcium channels (VGCCs), andinternal calcium mobilization from 1, 4, 5-trisphosphate (IP3)-sensitive stores andcaffeine/ryanodine-sensitive stores. In addition, a calcium-triggered exocytosismechanism was involved in the ES-induced NGF release from cultured Schwanncells.Conclusion: ES is capable of regulating the biological behaviors of Schwanncells, including proliferation, cell adhesion, spreading, as well as the expressionand secretion of NGF and BDNF. The regulatory effect of ES on Schwann cellmight partially contribute to the beneficial effect of ES on nerve regeneration.