Proximal Nerve Stump Research Articles

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.

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.

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.

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.

A Surgical Approach to Treat Painful Neuromas of the Supraorbital and Supratrochlear Nerves with Implantation of the Proximal Stump into the Orbit.

Frontal neuralgia causally related to trauma to the supraorbital and supratrochlear nerves remains a difficult problem to resolve. A peripheral nerve approach to this problem would involve neuroma resection and relocation of the proximal nerve stump to a location away from the vulnerable supraorbital ridge. A retrospective chart review was done to identify patients with frontal pain related to supraorbital trauma who underwent operative interventions to solve this problem by neuroma resection and relocation of the proximal stumps into the orbit. Eight patients were identified for inclusion in this study. At a mean of 16 months after surgery, there was a significant change in the visual analog score from a mean of 9.4 to 2.8 ( p < 0.05), with 88% of the patients reporting a >50% reduction in pain postoperatively. There was one treatment failure. There were no postoperative complications. The strategy of relocating the proximal end of the supraorbital and supratrochlear nerves into the posterior orbit after resecting the painful neuromas can successfully manage posttraumatic craniofacial pain related to these injured nerves.

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.

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration.

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury; however, the potential for full repair following a transection injury is much less. Currently, the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps, which prevent regenerating axons reaching the distal nerve. Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins, Slits, Ephrins and Semaphorins. Several recent studies have indicated key roles of Netrin1, Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury. Inside the nerve bridge, nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2. EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump. Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1; within Schwann cells, Robo1 expression is also Sox2-dependent. Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge. In addition to the Slit3/Robo1 signalling system, migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor. It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap. Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps. Therefore, understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.

Brief Electrical Stimulation Improves Functional Recovery After Femoral But Not After Facial Nerve Injury in Rats

Brief low-frequency electrical stimulation (ES, 1 h, 20 Hz) of the proximal nerve stump has emerged as a potential adjunct treatment for nerve injury. Despite available experimental and clinical data, the potentials and limitations of the ES therapy still have to be defined using different animal models, types of nerves, and clinical settings. Here, we show that brief ES of the proximal stump of the transected rat femoral nerve causes, as estimated by motion analysis, enhanced functional recovery reaching preoperative levels within 5 months of injury, in contrast to the incomplete restoration in sham-stimulated (SS) animals. The functional advantage seen in ES rats was associated with higher numbers, as compared with SS, of correctly targeted quadriceps motoneurons. In contrast, ES prior to facial nerve suture did not lead to improvement of whisking compared with SS. Lack of functional effects of the treatment was correlated with lack of changes, as compared with SS, in the precision of muscle reinnervation and frequency of abnormally innervated muscle fibers. These results show that ES is an effective therapy in a spinal nerve injury model leading to complete restoration of function. Although this finding and the safety of the procedure are encouraging, the results for the facial nerve model suggest that brief ES may not be a universal treatment for nerve injuries. Anat Rec, 302:1304-1313, 2019. © 2019 Wiley Periodicals, Inc.

Three-Dimensional In vivo Magnetic Resonance Imaging (MRI) of Mouse Facial Nerve Regeneration.

MRI (magnetic resonance imaging) is an indispensable tool in the diagnosis of centrals nervous system (CNS) disorders such as spinal cord injury and multiple sclerosis (MS). In contrast, diagnosis of peripheral nerve injuries largely depends on clinical and electrophysiological parameters. Thus, currently MRI is not regularly used which in part is due to small nerve calibers and isointensity with surrounding tissue such as muscles. In this study we performed translational MRI research in mice to establish a novel MRI protocol visualizing intact and injured peripheral nerves in a non-invasive manner without contrast agents. With this protocol we were able to image even very small nerves and nerve branches such as the mouse facial nerve (diameter 100–300 μm) at highest spatial resolution. Analysis was performed in the same animal in a longitudinal study spanning 3 weeks after injury. Nerve injury caused hyperintense signal in T2-weighted images and an increase in nerve size of the proximal and distal nerve stumps were observed. Further hyperintense signal was observed in a bulb-like structure in the lesion site, which correlated histologically with the production of fibrotic tissue and immune cell infiltration. The longitudinal MR representation of the facial nerve lesions correlated well with physiological recovery of nerve function by quantifying whisker movement. In summary, we provide a novel protocol in rodents allowing for non-invasive, non-contrast agent enhanced, high-resolution MR imaging of small peripheral nerves longitudinally over several weeks. This protocol might further help to establish MRI as an important diagnostic and post-surgery follow-up tool to monitor peripheral nerve injuries in humans.

Abstract 136: Denervation Induced Degradation of Motor Endplates can be Delayed Using Local Supplementation of Agrin at the Time of Injury.

PURPOSE: Robust muscle atrophy and fibrosis are cited as the prime causes for the permanence of motor deficits following surgical intervention for a denervation injury. Following a peripheral nerve transection, regenerating axons traverse long distances to reinnervate their target organs. However, due to the slow rate of axonal regrowth and a large segmental defect, muscle atrophy and destabilization of the neuromuscular junction (NMJ) usually proceeds before reinnervation occurs. Agrin is characterized as an essential component of NMJ formation and synaptogenesis. Here, we utilize an in vivo agrin deficient mouse nerve denervation model to both characterize the morphology of agrin deficient endplates as well as the response of the NMJ to local delivery of agrin following a denervation event. We found the endplates of agrin-deficient mice in a more degraded states than the endplates of wildtype mice following denervation. We also found that local delivery of agrin following denervation serves to transiently improve motor endplate morphology. We thus demonstrate the efficacy of using a biomolecular therapeutic approach to maintain and preserve the denervated NMJ. MATERIALS AND METHODS: A denervation model was created in 6-week-old mice from WT and agrin deficient C57BL/6 strains by excising 10 mm right sciatic nerve segment from the mid-thigh of the mice and suturing the proximal nerve stump to the gluteal muscle with 9-0 suture so as to prevent regeneration. Agrin deficient mice were either injected with supplemental agrin or PBS as a control at the site of injury. The downstream denervated and contralateral control soleus, plantaris and gastrocnemius muscles were harvested for immunohistochemistry, cryo-sections with H&E staining, and quantitative western blots at the 1,2,4,8 and 16-week timepoints. Quantification of motor endplate morphology was done using Volocity 3-D image software. Quantification of muscle fiber diameter was done using ImageJ software. RESULTS: Fluorescence confocal imaging of harvested soleus muscles revealed that agrin supplemented animals retained superior motor endplate morphology over control animals in all timepoints. The average surface area of agrin supplemented endplates were significantly greater than control endplates in all analyzed timepoints (p <0.05) CONCLUSION: Highlighting the importance of agrin, we have shown that even a single supplemental dose of agrin delivered locally at the site of injury is effective in preserving motor endplates in denervated mice hindlimbs even at the latest time points. This is consistent with our previous work that detailed MMP3 knock down maintained agrin at the NMJ and thereby preserved the motor endplates so as to improve function recovery. Taken together, these current experiments support the strategy to preserve motor endplates so as to prolong the window of opportunity for surgical intervention after a traumatic nerve injury.

End-to-Side Nerve Repair: Current Concepts and Future Perspectives.

Peripheral nerves injuries are extremely debilitating and have been a perennial challenge to the reconstructive surgeon. End-to-side (ETS) neurorrhaphy is a potential strategy for treating nerve lesions without usable proximal nerve stump. A number of interesting clinical and experimental studies have been carried out on ETS nerve repair during the first years of the 20th century. End-to-side nerve repair was then almost abandoned for more than 50 years. The rediscovery of ETS nerve repair was attributed to the experimental work of Viterbo.Two mechanisms have been suggested for axonal regeneration: terminal and collateral sprouting. While in collateral sprouting axonal outgrowth occurs along the length of the uninjured axons, in terminal sprouting, axonal regeneration takes place at the distal most aspect of either injured or uninjured axons. Clinical experience with ETS nerve repair includes management of upper extremity nerve injury, facial reanimation, reconstruction after tumor ablation, and the prevention of neuroma formation.The aim of this review article was 2-fold: (1) overviewing the most interesting experimental investigations and clinical studies published so far; (2) calling for more basic research on ETS nerve repair indicating the several unanswered questions that this concept asks to the microsurgical community.

The synaptic receptor Lrp4 promotes peripheral nerve regeneration

Early during PNS regeneration, regenerating axons emerge from the proximal nerve stump, yet whether they extend simultaneously or whether pioneering axons establish a path for follower axons remains unknown. Moreover, the molecular mechanisms underlying robust regeneration are incompletely understood. Using live imaging, we demonstrate that in zebrafish pioneering axons establish a regenerative path for follower axons. We find this process requires the synaptic receptor lrp4, and in lrp4 mutants pioneers are unaffected while follower axons frequently stall at the injury gap, providing evidence for molecular diversity between pioneering and follower axons in regeneration. We demonstrate that Lrp4 promotes regeneration through an axon extrinsic mechanism and independent of membrane anchoring and MuSK co-receptor signaling essential for synaptic development. Finally, we show that Lrp4 coordinates the realignment of denervated Schwann cells with regenerating axons, consistent with a model by which Lrp4 is repurposed to promote sustained peripheral nerve regeneration via axon-glia interactions.

MiR-129 controls axonal regeneration via regulating insulin-like growth factor-1 in peripheral nerve injury

The microenvironment of peripheral nerve regeneration consists of multiple neurotrophic factors, adhesion molecules, and extracellular matrix molecules, secreted by unique glial cells in the peripheral nerve system (PNS)-Schwann cell (SCs). Following peripheral nerve injury (PNI), local IGF-1 production is upregulated in SCs and denervated muscle during axonal sprouting and regeneration. Regulation of IGF-1/IGF-1R signaling is considered as a potentially targeted therapy of PNI. We previously identified a group of novel miRNAs in proximal nerve following rat sciatic nerve transection. The present work focused on the role of miR-129 in regulation of IGF-1 signaling after sciatic nerve injury. The temporal change profile of the miR-129 expression was negatively correlated with the IGF-1 expression in proximal nerve stump and dorsal root ganglion (DRG) following sciatic nerve transection. An increased expression of miR-129 inhibited proliferation and migration of SCs, and axonal outgrowth of DRG neurons, which was inversely promoted by silencing of the miR-129 expression. The IGF-1 was identified as one of the multiple target genes of miR-129, which exerted negative regulation of IGF-1 by translational suppression. Moreover, knockdown of IGF-1 attenuated the promoting effects of miR-129 inhibitor on proliferation and migration of SCs, and neurite outgrowth of DRG neurons. Overall, our data indicated that miR-129 own the potential to regulate the proliferation and migration of SCs by targeting IGF-1, providing further insight into the regulatory role of miRNAs in peripheral nerve regeneration. The present work not only provides new insight into miR-129 regulation of peripheral nerve regeneration by robust phenotypic modulation of neural cells, but also opens a novel therapeutic window for PNI by mediating IGF-1 production. Our results may provide further experimental basis for translation of the molecular therapy into the clinic.

Cell Adhesion Molecule Close Homolog of L1 (CHL1) Guides the Regrowth of Regenerating Motor Axons and Regulates Synaptic Coverage of Motor Neurons.

The close homolog of L1 (CHL1) is a cell adhesion molecule involved in regulation of neuronal differentiation and survival, neurite outgrowth and axon guidance during development. In the mature nervous system, CHL1 regulates synaptic activity and plasticity. The aim of the present study was to evaluate the influence of CHL1 on peripheral nerve regeneration after trauma. Using the established model of mouse femoral nerve regeneration, CHL1 knock-out mice were investigated in comparison to the wild type littermates. First, non-injured mice of both genotypes were compared regarding the synaptic phenotypes in the corresponding spinal cord segment. While no differences in phenotypes were detectable in the femoral nerve, corresponding segments in the spinal cord were observed to differ in that inhibitory perisomatic innervation of motor neurons was increased in CHL1-deficient mice, and numbers of perisomatic cholinergic synapses on motor neuronal somata were reduced. Regarding the femoral nerve after injury, CHL1-deficient mice demonstrated preferential motor axon regrowth into the saphenous vs. quadriceps branch after nerve transection upstream of the nerve bifurcation by 8 weeks after transection, indicating decreased preferential motor re-innervation. Furthermore, in injured wild-type mice, enhanced CHL1 expression was observed in regenerating axons in the proximal nerve stump upstream of the bifurcation at days 1, 3, 5, 7 and 14, and in the distal stump at days 7 and 14 after injury, when compared to non-injured mice. Injury-related upregulation of CHL1 expression was more pronounced in axons than in Schwann cells. Despite a more pronounced capacity for preferential motor axon regrowth in wild-type vs. mutant mice, only a tendency for difference in recovery of motor functions was observed between genotypes, without statistical significance Taken together, these results indicate that CHL1 is involved in peripheral nerve regeneration, because it guides regrowing axons into the appropriate nerve branch and regulates synaptic coverage in the spinal cord.

Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats.

Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (∼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.

Novel experimental surgical strategy to prevent traumatic neuroma formation by combining a 3D-printed Y-tube with an autograft.

OBJECTIVE Traumatic neuromas may develop after nerve injury at the proximal nerve stump, which can lead to neuropathic pain. These neuromas are often resistant to therapy, and excision of the neuroma frequently leads to recurrence. In this study, the authors present a novel surgical strategy to prevent neuroma formation based on the principle of centro-central anastomosis (CCA), but rather than directly connecting the nerve ends to an autograft, they created a loop using a 3D-printed polyethylene Y-shaped conduit with an autograft in the distal outlets. METHODS The 3D-printed Y-tube with autograft was investigated in a model of rat sciatic nerve transection in which the Y-tube was placed on the proximal sciatic nerve stump and a peroneal graft was placed between the distal outlets of the Y-tube to form a closed loop. This model was compared with a CCA model, in which a loop was created between the proximal tibial and peroneal nerves with a peroneal autograft. Additional control groups consisted of the closed Y-tube and the extended-arm Y-tube. Results were analyzed at 12 weeks of survival using nerve morphometry for the occurrence of neuroma formation and axonal regeneration in plastic semi-thin sections. RESULTS Among the different surgical groups, the Y-tube with interposed autograft was the only model that did not result in neuroma formation at 12 weeks of survival. In addition, a 13% reduction in the number of myelinated axons regenerating through the interposed autograft was observed in the Y-tube with autograft model. In the CCA model, the authors also observed a decrease of 17% in the number of myelinated axons, but neuroma formation was present in this model. The closed Y-tube resulted in minimal nerve regeneration inside the tube together with extensive neuroma formation before the entrance of the tube. The extended-arm Y-tube model clearly showed that the majority of the regenerating axons merged into the Y-tube arm, which was connected to the autograft, leaving the extended plastic arm almost empty. CONCLUSIONS This pilot study shows that our novel 3D-printed Y-tube model with interposed autograft prevents neuroma formation, making this a promising surgical tool for the management of traumatic neuromas.

Epineural Sheath Jacket as a New Surgical Technique for Neuroma Prevention in the Rat Sciatic Nerve Model

Terminal neuromas resulting from severe nerve injuries and traumatic or surgical limb amputations can become a source of pain, and significantly impair patients' quality of life. Recently, the number of patients with peripheral nerve injuries increased due to modern war conflicts, natural disasters, and traffic accidents. This study investigated the efficacy of the epineural sheath jacket (ESJ) as a novel technique for neuroma prevention in the rat sciatic nerve model. A 20-mm segment of the right sciatic nerve was excised in 18 Lewis rats, and the animals were divided into 3 experimental groups (n = 6/group): group I-control, nerve stump without protection; group II-muscle burying group, nerve stump buried in the muscle; group III-ESJ group, nerve stump protected by ESJ. The ESJ was created from the excised sciatic nerve and applied as a "cap" over the proximal nerve stump. The presence of neuropathic pain was assessed weekly by pinprick test and Tinel sign, up to 24 weeks postsurgery. At 24 weeks, assessments, such as macroscopic evaluation, retrograde neuronal labeling analysis, histomorphometry, and neural/connective tissue ratio were performed. Epineural sheath jacket significantly reduced neuroma formation, which was associated with decreased Tinel sign (16.7%, P < 0.05) response compared with the nerve stump control. Moreover, ESJ reduced axonal sprouting, bulb-shaped nerve ending formation and perineural adhesions, as confirmed by macroscopic evaluation. Histological evaluation confirmed that nerve stumps protected with the ESJ showed less fibrosis and presented well-organized axonal structure. Neural/connective tissue ratio and retrograde neuronal labeling analysis revealed significantly improved results in the ESJ group compared to the control nerve stump group (P = 0.032 and P = 0.042, respectively). The protective effect of the ESJ against neuroma formation was confirmed by behavioral and histological analyses, showing outcomes comparable to the muscle burying technique-the criterion standard of neuroma management.

Influence of Blood Flow on the Neuroma Formation after Transposition of the Nerve Stump into Vein: Experimental and Clinical Study.

We study the influence of blood flow on the neuroma formation after transposition of the nerve stump into a vein. A total of 30 rats were divided into two groups. In group (I), a 10-mm segment of the femoral vein was excluded from the blood stream. A venotomy was performed in the middle of this segment. The proximal nerve stump of femoral nerve was transposed into the lumen of the femoral vein via a venotomy and held with an epineural suture through the venous wall. In group (II), the same technique was used as in group (I) but without exclusion of the vein segment from blood flow. The control side of the femoral nerve was transected and then was left in place. All animals were sacrificed after 28 weeks. Histopathological evaluation was performed postoperatively; serial transverse sections were made to find the largest diameter of the neuroma. Selected specimens were processed for electron microscopy examination. The following parameters were assessed in both the groups: cross-sectional area of neuromas; myelinated axons; and neural tissue to connective tissue ratio. In group (I), the proximal end of the tibial nerve formed a nonclassic neuroma. In group (II), the proximal end of the tibial nerve formed a nonclassic neuroma smaller in diameter than group (I). The proximal end of the femoral nerve of the control sides in both groups formed a classic neuroma larger in diameter than the experimental sides in both the groups. A Kruskal-Wallis H test showed that there was a statistically significant difference in myelinated axons between the different groups, X2 (3) = 36.19, p < 0.001. The average neural tissue to connective tissue ratio in the control group (I) ranged from 39.2 to 85.8%, but on the experimental side, it was more or less equalized. The average of a neural tissue to connective tissue ratio of the group (II) ranged from 59.1 to 63.9% in the treatment sides as compared to 28.6 to 82.4% in the control sides. The clinical experience utilizing the same technique in the treatment of 10 patients of painful neuromas of the superficial radial nerve and digital nerves were presented with encouraging results. The blood flow has a considerable effect on neuroma formation. We recommend a further study to know the fine details about the exact role of blood as the bloodstream may carry away neurotrophic factors that may also be released by the nerve stump itself.