Distal Nerve Stump Research Articles

Elastin-like Proteins to Support Peripheral Nerve Regeneration in Guidance Conduits.

Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.

Regeneration of the peripheral nerve via multifunctional electrospun scaffolds.

Over the last two decades, electrospun scaffolds have proved to be advantageous in the field of nerve tissue regeneration by connecting the cavity among the proximal and distal nerve stumps growth cones and leading to functional recovery after injury. Multifunctional nanofibrous structure of these scaffolds provides enormous potential by combining the advantages of nano-scale topography, and biological science. In these structures, selecting the appropriate materials, designing an optimized structure, modifying the surface to enhance biological functions and neurotrophic factors loading, and native cell-like stem cells should be considered as the essential factors. In this systematic review paper, the fabrication methods for the preparation of aligned nanofibrous scaffolds in yarn or conduit architecture are reviewed. Subsequently, the utilized polymeric materials, including natural, synthetic and blend are presented. Finally, their surface modification techniques, as well as, the recent advances and outcomes of the scaffolds, both in vitro and in vivo, are reviewed and discussed.

Intravitreal Co-Administration of GDNF and CNTF Confers Synergistic and Long-Lasting Protection against Injury-Induced Cell Death of Retinal Ganglion Cells in Mice

We have recently demonstrated that neural stem cell-based intravitreal co-administration of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) confers profound protection to injured retinal ganglion cells (RGCs) in a mouse optic nerve crush model, resulting in the survival of ~38% RGCs two months after the nerve lesion. Here, we analyzed whether this neuroprotective effect is long-lasting and studied the impact of the pronounced RGC rescue on axonal regeneration. To this aim, we co-injected a GDNF- and a CNTF-overexpressing neural stem cell line into the vitreous cavity of adult mice one day after an optic nerve crush and determined the number of surviving RGCs 4, 6 and 8 months after the lesion. Remarkably, we found no significant decrease in the number of surviving RGCs between the successive analysis time points, indicating that the combined administration of GDNF and CNTF conferred lifelong protection to injured RGCs. While the simultaneous administration of GDNF and CNTF stimulated pronounced intraretinal axon growth when compared to retinas treated with either factor alone, numbers of regenerating axons in the distal optic nerve stumps were similar in animals co-treated with both factors and animals treated with CNTF only.

Migrating Schwann cells direct axon regeneration within the peripheral nerve bridge.

Schwann cells within the peripheral nervous system possess a remarkable regenerative potential. Current research shows that peripheral nerve-associated Schwann cells possess the capacity to promote repair of multiple tissues including peripheral nerve gap bridging, skin wound healing, digit tip repair as well as tooth regeneration. One of the key features of the specialized repair Schwann cells is that they become highly motile. They not only migrate into the area of damaged tissue and become a key component of regenerating tissue but also secrete signaling molecules to attract macrophages, support neuronal survival, promote axonal regrowth, activate local mesenchymal stem cells, and interact with other cell types. Currently, the importance of migratory Schwann cells in tissue regeneration is most evident in the case of a peripheral nerve transection injury. Following nerve transection, Schwann cells from both proximal and distal nerve stumps migrate into the nerve bridge and form Schwann cell cords to guide axon regeneration. The formation of Schwann cell cords in the nerve bridge is key to successful peripheral nerve repair following transection injury. In this review, we first examine nerve bridge formation and the behavior of Schwann cell migration in the nerve bridge, and then discuss how migrating Schwann cells direct regenerating axons into the distal nerve. We also review the current understanding of signals that could activate Schwann cell migration and signals that Schwann cells utilize to direct axon regeneration. Understanding the molecular mechanism of Schwann cell migration could potentially offer new therapeutic strategies for peripheral nerve repair.

Intercostal nerve transfer for restoration of the diaphragm muscle function after phrenic nerve transfer in total brachial plexus avulsion

ObjectTo determine the possibility of innervation of the diaphragm muscle using intercostal nerve after ipsilateral phrenic nerve transfer in total brachial plexus avulsion. MethodsBilateral phrenic nerves and the 9th intercostal nerves were observed inside the thorax. The point where the phrenic nerve entered the diaphragm muscle (point A), the point where the 9th intercostal nerve gave rise to the cutaneous branch (point B) and crossed the posterior axillary line (point C) and the point where the posterior axillary line met the insertion of the diaphragm muscle (point D) were identified. The distances between points B and C, points A and C and from points A through D to C were recorded respectively. The 9th intercostal nerve was transferred to the distal stump of the phrenic nerve in one patient after phrenic nerve transfer to avulsed brachial plexus. ResultsThe mean distances between points B and C, points A and C and from points A through D to C were 12.20 ± 1.04 cm, 10.32 ± 1.02 cm and 16.43 ± 0.91 cm on the right side respectively, 11.78 ± 1.21 cm, 7.77 ± 0.85 cm and 11.74 ± 1.00 cm on the left side respectively. The 9th intercostal nerve was used to innervate the distal stump of the phrenic nerve in one patient after the phrenic nerve transfer to the avulsed brachial plexus. The diaphragm muscle function partially recovered one year after the operation. ConclusionThe 9th intercostal nerve can be transferred to the distal stump of the phrenic nerve to restore the diaphragm muscle function according to the anatomical study. The movement of the diaphragm muscle was partially restored in one clinical case.

Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Transection is Driven by Cellular Intravitreal Sciatic Nerve Grafts.

Neurotrophic factors (NTF) secreted by Schwann cells in a sciatic nerve (SN) graft promote retinal ganglion cell (RGC) axon regeneration after either transplantation into the vitreous body of the eye or anastomosis to the distal stump of a transected optic nerve. In this study, we investigated the neuroprotective and growth stimulatory properties of SN grafts in which Schwann cells had been killed (acellular SN grafts, ASN) or remained intact (cellular SN grafts, CSN). We report that both intravitreal (ivit) implanted and optic nerve anastomosed CSN promote RGC survival and when simultaneously placed in both sites, they exert additive RGC neuroprotection. CSN and ASN were rich in myelin-associated glycoprotein (MAG) and axon growth-inhibitory ligand common to both the central nervous system (CNS) and peripheral nervous system (PNS) myelin. The penetration of the few RGC axons regenerating into an ASN at an optic nerve transection (ONT) site is limited into the proximal perilesion area, but is increased >2-fold after ivit CSN implantation and increased 5-fold into a CSN optic nerve graft after ivit CSN implantation, potentiated by growth disinhibition through the regulated intramembranous proteolysis (RIP) of p75NTR (the signalling trans-membrane moiety of the nogo-66 trimeric receptor that binds MAG and associated suppression of RhoGTP). Mϋller cells/astrocytes become reactive after all treatments and maximally after simultaneous ivit and optic nerve CSN/ASN grafting. We conclude that simultaneous ivit CSN plus optic nerve CSN support promotes significant RGC survival and axon regeneration into CSN optic nerve grafts, despite being rich in axon growth inhibitory molecules. RGC axon regeneration is probably facilitated through RIP of p75NTR, which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts.

Transfert du nerf obturateur vers le nerf tibial au moyen d’une greffe de nerf saphène pour le traitement des avulsions du plexus sacré : étude cadavérique

IntroductionIn cases of sacral nerve avulsion injuries, proximal nerve stumps are not available because of its protected position in pelvis, and results of nerve repair or graft are unsatisfactory. Nerve transfer can reduce the regeneration time and improve the results of recovery. HypothesisThe obturator nerve transfer to the tibial nerve via saphenous nerve graft is possible and feasible. Materials and methodsTen male adult cadaveric lower limbs dissected to identify the location of the anterior branch of obturator nerve, the saphenous nerve and the medial gastrocnemius branch. The saphenous nerve was cut from its origin and transferred to the anterior branch of obturator nerve. As well, it was cut distally and transferred to the medial gastrocnemius branch. After nerve coaptation, surface area and fascicle count were determined by histological methods. ResultsIn all limbs, the proximal and distal stumps of saphenous nerve were reached the anterior branch of obturator and the medial gastrocnemius branch, respectively without tension. The mean of fascicle number in the anterior branch of obturator nerve, proximal and distal stump of the saphenous nerve and stump of medial gastrocnemius nerve branch were 2.90±0.99, 4.50±2.70, 4.00±2.26 and 4.30±1.25, respectively. DiscussionThis study showed that it is possible to transfer the obturator nerve to the medial gastrocnemius branch via saphenous nerve bridge; and their histological parameters are match in a good manner. Therefore, this technique is suggested for patients with sacral nerve avulsion injuries. Level of evidenceIV, case series of cadaveric study.

Obturator to tibial nerve transfer via saphenous nerve graft for treatment of sacral plexus root avulsions: A cadaveric study

IntroductionIn cases of sacral nerve avulsion injuries, proximal nerve stumps are not available because of its protected position in pelvis, and results of nerve repair or graft are unsatisfactory. Nerve transfer can reduce the regeneration time and improve the results of recovery. HypothesisThe obturator nerve transfer to the tibial nerve via saphenous nerve graft is possible and feasible. Materials and methodsTen male adult cadaveric lower limbs dissected to identify the location of the anterior branch of obturator nerve, the saphenous nerve and the medial gastrocnemius branch. The saphenous nerve was cut from its origin and transferred to the anterior branch of obturator nerve. As well, it was cut distally and transferred to the medial gastrocnemius branch. After nerve coaptation, surface area and fascicle count were determined by histological methods. ResultsIn all limbs, the proximal and distal stumps of saphenous nerve were reached the anterior branch of obturator and the medial gastrocnemius branch, respectively without tension. The mean of fascicle number in the anterior branch of obturator nerve, proximal and distal stump of the saphenous nerve and stump of medial gastrocnemius nerve branch were 2.90±0.99, 4.50±2.70, 4.00±2.26 and 4.30±1.25, respectively. DiscussionThis study showed that it is possible to transfer the obturator nerve to the medial gastrocnemius branch via saphenous nerve bridge; and their histological parameters are match in a good manner. Therefore, this technique is suggested for patients with sacral nerve avulsion injuries. Level of evidenceIV, case series of cadaveric study.

Stem cells in end-to-side neurorrhaphy. Experimental study in rats.

Purpose:To evaluate the influence of mesenchymal stem cells from adipose tissue in the end-to-side neurorrhaphy, focusing in the nerve regeneration and the muscle reinnervation in acute trauma.Methods:140 animals were randomly divided in seven groups: control, denervated, end-to-side neurorrhaphy between distal stump of common peroneal nerve and tibial nerve (ESN), ESN wrapped in fascia, ESN wrapped in fascia and platelet gel, ESN wrapped in platelet gel, ESN wrapped in fascia and platelet gel within stem cells (without culture) removed from the adipose tissue. Mass measurements of the animal and of cranial tibial muscles, electromyography, walking track analysis tests and histological examinations of the nerves and muscles after 180 days was performed.Results:In the groups where the ESN was performed, the results were always better when compared to the denervated group, showing reinnervation in all ESN groups. The most sensitive methods were walking track and histological analysis. Only the group with stem cells showed values similar to the control group, as well as the functional indices of peroneal nerve and the number of nerve fibers in the peroneal nerve.Conclusions:Stem cells were effective in ESN according with the functional index of the peroneal nerve, evaluated by walking track analysis and the number of nerve fibers in the peroneal nerve.

LONG-TERM OUTCOMES OF RAT SCIATIC NERVE INJURY AND ITS GRAFTING WITH SILICON WIRES-BASED CONDUIT: DISTAL NERVE STUMP HISTOLOGY AND ELECTROMYOGRAPHY RESULTS

Relevance. The investigations on trauma epidemiology have shown that both combat- and noncombat-related extremity injuries are often accompanied by nerve injuries. These injuries disproportionately affect young healthy civilians and military officers and has a devastating impact on a patients’ quality of life.
 Severe nerve injuries, such as nerve trunk injury in continuity (Sunderland 5), that cannot be treated by neurorraphy without tension, require use of nerve gap bridging strategies with different materials and techniques.
 Objective. This study was aimed to evaluate any positive or negative impact of implanted silicon wires on the quality of nerve fibers at distal nerve stump.
 Materials and Methods. An experiment was performed on 40 male Whistar rats 2-4 month that were divided to the next groups:
 I, (n=10) sham-operated, only surgical access to sciatic nerve was performed.
 II (n=10) with 10 mm sciatic nerve gap that was bridged with autoneurografting.
 III (n=10) with 10 mm nerve gap that was bridged with allogenic decell aorta filled with 4% carboxymethylcellulose hydrogel.
 IV (n=10) with 10 mm nerve gap that was bridged with allogenic decell aorta filled with 4% carboxymethylcellulose hydrogel and aligned p-type silicon microvires.
 Decellularization of allogenic aortas was performed by freeze-thaw cycles.
 Silicon whiskers were fabricated by Vapor-Liquid-Solid (VLS) method in a cold wall Catalytic Chemical Vapor Deposition (Cat-CVD) chamber, pre-cleaned with hydrofluoric acid and sterilized via 180*C dry heat.
 12 weeks after surgery under general anesthesia all rats underwent invasive needle electroneurpmyography with proximal nerve stump stimulation and registration from gastrocnemius muscle. Myograms were recorded and compared by the shape of M-reflex and its amplitude.
 After myography rats were euthanized under thiopentone overdosage and distal stumps of injured sciatic nerves were harvested for light microscopy.
 Sciatic nerve transverse slices were stained with nitric silver by modified Bielschowsky method Nerve fiber diameter, axon diameter, myelin sheath thickness and axon-to-nerve fiber diameter ratio (g-ratio) were measured.
 Results. Performed analysis showed that rats from ІІ and IV groups demonstrated the best quality of nerve fibers in distal nerve stump. That was evidenced by bigger nerve fibers diameter in rats from autologous nerve grafting group and aorta with gel and wires grafting group in comparison with aorta with gel grafting group. Rats from IV demonstrated higher voltage and lower latency of M-reflexes during electromyography.
 Conclusions. It can be concluded about the possible pro-regenerative impact of implanted silicon wires that was evidenced by better nerve fibers quality at distal nerve stump.

Distinct VIP and PACAP Functions in the Distal Nerve Stump During Peripheral Nerve Regeneration

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Peptide (PACAP) are regeneration-associated neuropeptides, which are up-regulated by neurons following peripheral nerve injury. So far, they have only been studied for their roles as autocrine signals for both neuronal survival and axon outgrowth during peripheral nerve regeneration. In this report, we examined VIP and PACAP’s paracrine effects on Schwann cells and macrophages in the distal nerve stump during peripheral nerve regeneration. We show that VPAC1, VPAC2, and PAC1 are all up-regulated in the mouse distal nerve following peripheral nerve injury and are highly expressed in Schwann cells and macrophages within the distal sciatic nerve. We further investigated the effect of VIP and PACAP on cultured rat Schwann cells, and found that VIP and PACAP can not only promote myelin gene expression in Schwann cells but can also inhibit the release of pro-inflammatory cytokines by Schwann cells. Furthermore, we show that VIP and PACAP inhibit the release of pro-inflammatory cytokines and enhance anti-inflammatory cytokine expression in sciatic nerve explants. Our results provide evidence that VIP and PACAP could have important functions in the distal nerve stump following injury to promote remyelination and regulate the inflammatory response. Thus, VIP and PACAP receptors appear as important targets to promote peripheral nerve repair following injury.

Analysis of Schwann Cell Migration and Axon Regeneration Following Nerve Injury in the Sciatic Nerve Bridge.

While it is proposed that interaction between Schwann cells and axons is key for successful nerve regeneration, the behavior of Schwann cells migrating into a nerve gap following a transection injury and how migrating Schwann cells interact with regenerating axons within the nerve bridge has not been studied in detail. In this study, we combine the use of our whole-mount sciatic nerve staining with the use of a proteolipid protein-green fluorescent protein (PLP-GFP) mouse model to mark Schwann cells and have examined the behavior of migrating Schwann cells and regenerating axons in the sciatic nerve gap following a nerve transection injury. We show here that Schwann cell migration from both nerve stumps starts later than the regrowth of axons from the proximal nerve stump. The first migrating Schwann cells are only observed 4 days following mouse sciatic nerve transection injury. Schwann cells migrating from the proximal nerve stump overtake regenerating axons on day 5 and form Schwann cell cords within the nerve bridge by 7 days post-transection injury. Regenerating axons begin to attach to migrating Schwann cells on day 6 and then follow their trajectory navigating across the nerve gap. We also observe that Schwann cell cords in the nerve bridge are not wide enough to guide all the regenerating axons across the nerve bridge, resulting in regenerating axons growing along the outside of both proximal and distal nerve stumps. From this analysis, we demonstrate that Schwann cells play a crucial role in controlling the directionality and speed of axon regeneration across the nerve gap. We also demonstrate that the use of the PLP-GFP mouse model labeling Schwann cells together with the whole sciatic nerve axon staining technique is a useful research model to study the process of peripheral nerve regeneration.

Reactivation of Denervated Schwann Cells by Embryonic Spinal Cord Neurons to Promote Axon Regeneration and Remyelination.

In peripheral nerve injuries (PNIs) in which proximal axons do not regenerate quickly enough, significant chronic degeneration of Schwann cells (SCs) can occur at the distal stump of the injured nerve and obstruct regeneration. Cell transplantation can delay the degeneration of SCs, but transplanted cells fail to generate voluntary electrical impulses without downstream signal stimulation from the central nervous system. In this study, we combined cell transplantation and nerve transfer strategies to investigate whether the transplantation of embryonic spinal cord cells could benefit the microenvironment of the distal stump of the injured nerve. The experiment consisted of two stages. In the first-stage surgery, common peroneal nerves were transected, and embryonic day 14 (E14) cells or cell culture medium was transplanted into the distal stump of the CPs. Six months after the first-stage surgery, the transplanted cells were removed, and the nerve segment distal to the transplanted site was used to bridge freshly cut tibial nerves to detect whether the cell-treated graft promoted axon growth. The phenotypic changes and the neurotrophic factor expression pattern of SCs distal to the transplanted site were detected at several time points after cell transplantation and excision. The results showed that at different times after transplantation, the cells could survive and generate neurons. Thus, the neurons play the role of proximal axons to prevent chronic degeneration and fibrosis of SCs. After excision of the transplanted cells, the SCs returned to their dedifferentiated phenotype and upregulated growth-associated gene expression. The ability of SCs to be activated again allowed a favorable microenvironment to be created and enhanced the regeneration and remyelination of proximal axons. Muscle reinnervation was also elevated. This transplantation strategy could provide a treatment option for complex neurological injuries in the clinic.

Improving corneal nerve regeneration: initial results of a novel regenerative agent

AbstractIt is well stablished that nerve deficit in the cornea leads to an imbalance in trophic factors required to maintain a healthy corneal tissue. This deficit is clinically manifested as impaired epithelial repair and wound healing, and eventually stromal scarring and ulceration, leading to a greater risk of transplant failure. Thus, corneal trophic environment must be restored by first promoting an adequate corneal nerve regeneration after transplant, although nowadays there is no approved neuro‐regenerative therapy for the cornea.A plethora of cytokines, chemokines and growth factors influence neuron survival and axon regeneration after lesion, most of them produced locally by Schwann cells at the distal nerve stump after nerve axotomy. In ARREST BLINDNESS work package 7, the efficacy of topical treatment with several cytokines, chemokines and trophic factors to promote corneal axon regeneration and the restoration of corneal sensory and trophic functions has been tested in an experimental model of surgically‐induced neurotrophic deficit, induced by performing a corneal stromal flap in the guinea‐pig. Using NGF as a positive control, protein neublastin, a member of the glial cell line derived neurotrophic factor family has been identified as the most effective factor to increase nerve regeneration after corneal lesion.(Supported by ARREST BLINDNESS, a project funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 667400)

Electrical synapses interconnecting axons in the optic nerve head–a novel model of optic nerve function

AbstractPurposeAxons in the optic nerve are arranged in bundles and conducting action potential with resistance related to their membrane. Here, we aim to characterize the function of newly identified gap junctions coupling optic nerve axons with each other’s.MethodsProximal and distal rat optic nerve stumps were processed for immunostainings, electron microscopy (EM), RT‐PCR and western blots (WB). Additional 20 animals were deeply anesthetized, and stainless‐steel screw electrodes were drilled in skulls in standard locations to record fVEPs. Recordings were performed in untouched animals, after retrobulbar injection of saline (negative control) or meclofenamic acid solution (gap junctions’ blocker). Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research, University of Toronto, Canada.ResultsImmunostaining of rat and human samples revealed presence of Cx45 and 36 in axonal membranes, colocalizing with β3‐tubulin, but not with GFAP. In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 ‐ proximal stump; 0.44 ± 0.02 ‐ distal stump), Cx45 showed higher levels (0.68 ± 0.0003 ‐ proximal stump; 0.9 ± 0.07 ‐ distal stump). Additionally, we detected transcript of Cx45 and Cx36 in RT‐PCR analysis of optic nerve homogenates. In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) that are directly coupling neighboring axons. In fVEPs, blocking of gap junctions’ with meclofenamic acid resulted in prolongation of the latency of P1 wave up to 140% after 5 min from retrobulbar injection of this blocker (p < 0.001) and 170% after 30 min (p < 0.001).ConclusionsOptic nerve axons do not form absolutely independent conductive channels. They are directly coupled by gap junctions formed in majority by neuronal Cx45. Coupling of axons allows to reduce axonal membrane resistance and accelerates transduction of visual signal.

Genetic changes in rat proximal nerve stumps after sciatic nerve transection.

Peripheral nerves can self-regenerate after traumatic injury, although their self-regeneration ability is limited after severe nerve injury. After peripheral nerve injury, the distal nerve stumps undergo Wallerian degeneration while the proximal nerve stumps undergo a regeneration process. Here, to decipher genetic changes and involved biological processes in the proximal nerve stumps after peripheral nerve injury, microarray data (GSE30165) were analyzed. Differentially expressed genes in the proximal nerve stumps at 0.5 h, 1 h, 3 h, 6 h, 9 h, 1 d, 4 d, 7 d, and 14 d after rat sciatic nerve transection were subjected to Ingenuity pathway analysis (IPA) bioinformatic analysis. Cytokine signaling, cellular immune response, nuclear receptor signaling, disease-specific pathways, and organismal growth and development were significantly activated in the proximal nerve stumps after nerve transection. Organ development, inflammation and immune response, diseases and organ abnormalities, and cellular behavior-related biological functions were highly involved. The expression levels of differentially expressed genes in biological function "Organismal Injury and Abnormalities" were displayed and validated. Our current study helps to obtain a better understanding of the biological processes of peripheral nerve regeneration, especially the regeneration process in the proximal nerve stumps, and thus may help to discover new therapeutic methods that can promote nerve regeneration.

Electrical Muscle Stimulation Accelerates Functional Recovery After Nerve Injury

Electrical muscle stimulation has been demonstrated to facilitate nerve regeneration and functional recovery, but the underlying mechanism remains only partially understood. In this study, we investigated the positive effect of electrical muscle stimulation following nerve injury and its molecular mechanisms of autophagy regulation. The sciatic nerves of Sprague-Dawley rats were transected and immediately repaired. Gastrocnemius muscles were electrically stimulated using surface electrodes. Motor functional recovery was assessed by gait analysis, nerve conduction examination and histological appearance of the target muscle. Axon regeneration was investigated by morphometric analysis. Western blotting and immunofluorescence staining were used to detect the expression of molecular biological changes in distal nerve stump. Ultrastructural features of the nerve were evaluated by transmission electron microscope. We found that axon regeneration and motor functional recovery were improved by electrical muscle stimulation. The number of autophagosomes and the expression of autophagy marker LC3-Ⅱ in distal nerve stump were increased while the level of autophagy substrate protein P62 was decreased following electrical muscle stimulation. Blockage of the autophagy flux by chloroquine (CQ) diminished the positive effect of electrical muscle stimulation on nerve injury. These results illustrated that electrical muscle stimulation accelerates axon regeneration and functional recovery through promoting autophagy flux in distal nerve segments following nerve injury and immediate repair (IR) by a so far unknown mechanism.

Dynamic expression of Slit1-3 and Robo1-2 in the mouse peripheral nervous system after injury.

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors. Although we previously reported that Slit1-3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system, how this expression changes after injury has not been well studied. Herein, we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve. At 14 days after injury, quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1-3 and Robo1-2 in L4-5 spinal cord and dorsal root ganglia, as well as the sciatic nerve. Immunohistochemical analysis was performed to examine Slit1-3, Robo1-2, neurofilament heavy chain, F4/80, and vimentin in L4-5 spinal cord, L4 dorsal root ganglia, and the sciatic nerve. Co-expression of Slit1-3 and Robo1-2 in L4 dorsal root ganglia was detected by in situ hybridization. In addition, Slit1-3 and Robo1-2 protein expression in L4-5 spinal cord, L4 dorsal root ganglia, and sciatic nerve were detected by western blot assay. The results showed no significant changes of Slit1-3 or Robo1-2 mRNA expression in the spinal cord within 14 days after injury. In the dorsal root ganglion, Slit1-3 and Robo1-2 mRNA expression were initially downregulated within 4 days after injury; however, Robo1-2 mRNA expression returned to the control level, while Slit1-3 mRNA expression remained upregulated during regeneration from 4-14 days after injury. In the sciatic nerve, Slit1-3 and their receptors Robo1-2 were all expressed in the proximal nerve stump; however, Slit1, Slit2, and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury. Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury. Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge. Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury. Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration, and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury. This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No. 30/3203) on April 12, 2014.

Investigation of axonal regeneration of Triturus ivanbureschi by using physiological and proteomic strategies

Peripheral nerve injuries are frequently observed and successful treatment depends mainly on the injury type, location of the damage, and the elapsed time prior to treatment. The regenerative capacity is limited only to the embryonic period in many mammalian tissues, but urodele amphibians do not lose this feature during adulthood. The main purpose of this study is to define the recovery period after serious sciatic nerve damage of a urodele amphibian, Triturus ivanbureschi. Experimental transection damage was performed on the sciatic nerves of T. ivanbureschi specimens. The recovery period of sciatic nerves were investigated by walking track analysis, electrophysiological recordings, and bottom-up proteomic strategies at different time points during a 35-day period. A total of 34 proteins were identified related to the nerve regeneration process. This study showed that the expression levels of certain proteins differ between distal and proximal nerve endings during the regeneration period. In distal nerve stumps, transport proteins, growth factors, signal, and regulatory molecules are highly expressed, whereas in proximal nerve stumps, neurite elongation proteins, and cytoskeletal proteins are highly expressed.

Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function.

PurposeTo characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head.MethodsTwenty‐five Wistar rats were killed by CO 2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 μ m meclofenamic acid solution (gap junctions’ blocker) were recorded. Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research.ResultsImmunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with β3‐tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001).ConclusionsOptic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.