Polyethylene glycol fusion repair of severed rat sciatic nerves reestablishes axonal continuity and reorganizes sensory terminal fields in the spinal cord

Peripheral nerve injuries result in the rapid degeneration of distal nerve segments and immediate loss of motor and sensory functions;behavioral recovery is typically poor.We used a plasmalemmal fusogen,polyethylene glycol(PEG),to immediately fuse closely apposed open ends of severed proximal and distal axons in rat sciatic nerves.We have previously reported that sciatic nerve axons repaired by PEG-fusion do not undergo Wallerian degeneration,and PEG-fused animals exhibit rapid(within 2–6 weeks)and extensive locomotor recovery.Furthermore,our previous report showed that PEG-fusion of severed sciatic motor axons was non-specific,i.e.,spinal motoneurons in PEG-fused animals were found to project to appropriate as well as inappropriate target muscles.In this study,we examined the consequences of PEG-fusion for sensory axons of the sciatic nerve.Young adult male and female rats(Sprague–Dawley)received either a unilateral single cut or ablation injury to the sciatic nerve and subsequent repair with or without(Negative Control)the application of PEG.Compound action potentials recorded immediately after PEG-fusion repair confirmed conduction across the injury site.The success of PEG-fusion was confirmed through Sciatic Functional Index testing with PEG-fused animals showing improvement in locomotor function beginning at 35 days postoperatively.At 2–42 days postoperatively,we anterogradely labeled sensory afferents from the dorsal aspect of the hindpaw following bilateral intradermal injection of wheat germ agglutinin conjugated horseradish peroxidase.PEG-fusion repair reestablished axonal continuity.Compared to unoperated animals,labeled sensory afferents ipsilateral to the injury in PEG-fused animals were found in the appropriate area of the dorsal horn,as well as inappropriate mediolateral and rostrocaudal areas.Unexpectedly,despite having intact peripheral nerves,similar reorganizations of labeled sensory afferents were also observed contralateral to the injury and repair.This central reorganization may contribute to the improved behavioral recovery seen after PEG-fusion repair,supporting the use of this novel repair methodology over currently available treatments.

A hyaluronic acid granular hydrogel nerve guidance conduit promotes regeneration and functional recovery of injured sciatic nerves in rats

A hyaluronic acid granular hydrogel can promote neuronal and astrocyte colony formation and axonal extension in vitro,suggesting that the hydrogel can simulate an extracellular matrix structure to promote neural regeneration.However,in vivo experiments have not been conducted.In this study,we transplanted a hyaluronic acid granular hydrogel nerve guidance conduit to repair a 10-mm long sciatic nerve gap.The Basso,Beattie,and Bresnahan locomotor rating scale,sciatic nerve compound muscle action potential recording,Fluoro-Gold retrograde tracing,growth related protein 43/S100 immunofluorescence staining,transmission electron microscopy,gastrocnemius muscle dry/wet weight ratio,and Masson’s trichrome staining results showed that the nerve guidance conduit exhibited similar regeneration of sciatic nerve axons and myelin sheath,and recovery of the electrophysiological function and motor function as autologous nerve transplantation.The conduit results were superior to those of a bulk hydrogel or silicone tube transplant.These findings suggest that tissue-engineered nerve conduits containing hyaluronic acid granular hydrogels effectively promote the morphological and functional recovery of the injured sciatic nerve.The nerve conduits have the potential as a material for repairing peripheral nerve defects.

Expression of NF-κB in Schwann cells and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in rats

Objective：To explore the expression of nuclear factor-kappa B （NF-kB） in Schwann cells （SCs） and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods： Thirty-six adult Sprague-Dawley （SD） rats were divided randomly into normal control group （n=6）, and sciatic nerves crushing group （n= 30）, and the later was further equally randomized into 5 subgroups： 1, 3, 7, 14, and 21 d post-injury groups. The expression of NF-kB of normal and injured nerves were examined by immunohistochemistry staining, and the apoptosis of motor neurons in spinal cord of lumbar 4 to lumbar 6 （L4-L6） was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling （TUNEL） assay. Both were qua.ntitated by image analysis. Results： In crushing group, except 21 d post-injury group, the expression of NF-kB was markedly higher than that in the normal control group （P〈0.05, P〈0. 01）. At 1 d after sciatic nerves crushing, the expression of NF-kB was obviously up-regulated, reached peak at 3 d, and recovered at 21 d. The same trend was observed in the time-course on motor neuron apoptosis after sciatic nerves injury. Correlation analyses revealed that motor neuron apoptosis was significantly and positively correlated with the expression of NF-kB following sciatic nerves injury （r= 0. 976 0, P〈0. 01）. Conclusion： After injury of sciatic nerves, the presence and up-regulation of NF-kB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.

Biomechanical properties of acellular sciatic nerves treated with a modified chemical method

Nerve grafts are able to adapt to surrounding biomechanical environments if the nerve graft itself exhibits appropriate biomechanical properties （load, elastic modulus, etc.）. The present study was designed to determine the differences in biomechanical properties between fresh and chemically acellularized sciatic nerve grafts. Two different chemical methods were used to establish acellular nerve grafts. The nerve was chemically extracted in the Sondell method with a combination of Triton X-100 （nonionic detergent） and sodium deoxycholate （anionic detergent）, and in the modified method with a combination of Triton X-200 （anionic detergent）, sulfobetaine-10 （SB-10, amphoteric detergents）, and sulfobetaine-16 （SB-16, amphoteric detergents）. Following acellularization, hematoxylin-eosin staining and scanning electron microscopy demonstrated that the effect of acellularization via the modified method was similar to the traditional Sondell method. However, effects of demyelination and nerve fiber tube integrity were superior to the traditional Sondell method. Biomechanical testing showed that peripheral nerve graft treated using the chemical method resulted in decreased biomechanical properties （ultimate load, ultimate stress, ultimate strain, and mechanical work to fracture） compared with fresh nerves, but the differences had no statistical significance （P 〉 0.05）. These results demonstrated no significant effect on biomechanical properties of nerves treated using the chemical method. In conclusion, nerve grafts treated via the modified method removed Schwann cells, preserved neural structures, and ensured biomechanical properties of the nerve graft, which could be more appropriate for implantation studies.

Galanin and its receptor system promote the repair of injured sciatic nerves in diabetic rats

Various studies have reported that galanin can promote axonal regeneration of dorsal root ganglion neurons in vitro and inhibit neuropathic pain. However, little is known about its effects on diabetic peripheral neuropathy, and in vivo experimental data are lacking. We hypothesized that repeated applications of exogenous galanin over an extended time frame may also repair nerve damage in diabetic peripheral neuropathy, and relieve pain in vivo. We found that neuropathic pain occurred in streptozotocin-induced diabetic rats and was more severe after sciatic nerve pinch injury at 14 and 28 days than in diabetic sham-operated rats. Treatment with exogenous galanin alleviated the neuropathic pain and promoted sciatic nerve regeneration more effectively in diabetic rats than in non-diabetic rats after sciatic nerve pinch injury. This was accompanied by changes in the levels of endogenous galanin, and its receptors galanin receptor 1 and galanin receptor 2 in the dorsal root ganglia and the spinal dorsal horn when compared with nerve pinch normal rats. Our results show that application of exogenous galanin daily for 28 days can promote the regeneration of injured sciatic nerves, and alleviate neuropathic pain in diabetic rats.

Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats

Human umbilical cord mesenchymal stem cells,incorporated into an amnion carrier tubes,were assessed for nerve regeneration potential in a rat nerve defect model.Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell （cell transplantation group）or saline（control group）.At 8,12,16 and 20 weeks after cell implantation,the sciatic functional index was higher in the cell transplantation group compared with the control group.Furthermore,electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased.Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time.Gastrocnemius muscle cell diameter,wet weight and restoration ratio were increased.These data indicate that transplanted human umbilical cord mesenchymal stem cells,using the amnion tube connection method,promote restoration of damaged sciatic nerves in rats.

Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves

Several studies have demonstrated that human umbilical cord blood-derived mesenchymal stem cells can promote neural regeneration following brain injury. However, the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells in guiding peripheral nerve regeneration remain poorly understood. This study was designed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells on neural regeneration using a rat sciatic nerve crush injury model. Human umbilical cord blood-derived mesenchymal stem cells （1 ~ 106） or a PBS control were injected into the crush-injured segment of the sciatic nerve. Four weeks after cell injection, brain-derived neurotrophic factor and tyrosine kinase receptor B mRNA expression at the lesion site was increased in comparison to control. Furthermore, sciatic function index, Fluoro Gold-labeled neuron counts and axon density were also significantly increased when compared with control. Our results indicate that human umbilical cord blood-derived mesenchvmal stem cells promote the functinnal r~.RcJv^rv nf P.n I^h-inillr^4 ~r^i~tit, n^r~e

Methylene blue enhances polyethylene glycol-fusion repair of completely severed rat sciatic nerves

Complete transection of peripheral mixed nerves immediately produces loss of sensory perception,muscle contractions and voluntary behavior mediated by the severed distal axons.In contrast to natural regeneration(~1 mm/d)of proximal axons that may eventually reinnervate denervated targets,re-innervation is restored within minutes by PEG-fusion that consists of neurorrhaphy and a sequence of well specified hypo-and isotonic calcium-free or calcium-containing solutions,the anti-oxidant methylene blue(MB)and the membrane fusogen polyethylene glycol(PEG).In this study,we examined the relative efficacy of PEG-fusion with no MB(0%),0.5%MB,or 1%MB on the recovery of voluntary behaviors by female Sprague-Dawley rats with a complete mid-thigh severance of their sciatic nerve bathed in extracellular fluid or calcium-containing isotonic saline.The recovery of voluntary behaviors is the most relevant measure of success of any technique to repair peripheral nerve injuries.We assessed recovery by the sciatic functional index,a commonly used measure of voluntary hindlimb behaviors following complete sciatic transections.We reported that both 1%MB and 0.5%MB in sterile distilled water in our PEG-fusion protocol with neurorrhaphy significantly increased the rate and extent of behavioral recovery compared to PEG plus neurorrhaphy alone.Furthermore,0.5%MB was as effective as 1%MB in voluntary behavioral recovery as assessed by the sciatic functional index.Since sterile 1%MB is no longer clinically available,we therefore recommend that 0.5%MB be included in upcoming human clinical trials to evaluate the safety and efficacy of PEG-fusion.All animal procedures were approved by the University of Texas Institutional Animal Care and Use Committee(AUP-2019-00225)on September 9,2020.

Electric stimulation and decimeter wave therapy improve the recovery of injured sciatic nerves

Drug treatment, electric stimulation and decimeter wave therapy have been shown to promote the repair and regeneration of the peripheral nerves at the injured site. This study prepared a Mackinnon’s model of rat sciatic nerve compression. Electric stimulation was given immediately after neurolysis, and decimeter wave radiation was performed at 1 and 12 weeks post-operation. Histological observation revealed that intraoperative electric stimulation and decimeter wave therapy could improve the local blood circulation of repaired sites, alleviate hypoxia of compressed nerves, and lessen adhesion of compressed nerves, thereby decreasing the formation of new entrapments and enhancing compressed nerve regeneration through an improved microenvironment for regeneration. Immunohistochemical staining results revealed that intraoperative electric stimulation and decimeter wave could promote the expression of S-100 protein. Motor nerve conduction velocity and amplitude, the number and diameter of myelinated nerve fibers, and sciatic functional index were significantly increased in the treated rats. These results verified that intraoperative electric stimulation and decimeter wave therapy contributed to the regeneration and the recovery of the functions in the compressed nerves.

Biodegradable magnesium wire promotes regeneration of compressed sciatic nerves

Magnesium（Mg） wire has been shown to be biodegradable and have anti-inflammatory properties. It can induce Schwann cells to secrete nerve growth factor and promote the regeneration of nerve axons after central nervous system injury. We hypothesized that biodegradable Mg wire may enhance compressed peripheral nerve regeneration. A rat acute sciatic nerve compression model was made, and AZ31 Mg wire（3 mm diameter; 8 mm length） bridged at both ends of the nerve. Our results demonstrate that sciatic functional index, nerve growth factor, p75 neurotrophin receptor, and tyrosine receptor kinase A m RNA expression are increased by Mg wire in Mg model. The numbers of cross section nerve fibers and regenerating axons were also increased. Sciatic nerve function was improved and the myelinated axon number was increased in injured sciatic nerve following Mg treatment. Immunofluorescence histopathology showed that there were increased vigorous axonal regeneration and myelin sheath coverage in injured sciatic nerve after Mg treatment. Our findings confirm that biodegradable Mg wire can promote the regeneration of acute compressed sciatic nerves.

EZH2-dependent myelination following sciatic nerve injury

Demyelination and remyelination have been major focal points in the study of peripheral nerve regeneration following peripheral nerve injury.Notably,the gene regulatory network of regenerated myelin differs from that of native myelin.Silencing of enhancer of zeste homolog 2(EZH2)hinders the differentiation,maturation,and myelination of Schwann cells in vitro.To further determine the role of EZH2 in myelination and recovery post-peripheral nerve injury,conditional knockout mice lacking Ezh2 in Schwann cells(Ezh2^(fl/fl);Dhh-Cre and Ezh2^(fl/fl);Mpz-Cre)were generated.Our results show that a significant proportion of axons in the sciatic nerve of Ezh2-depleted mice remain unmyelinated.This highlights the crucial role of Ezh2 in initiating Schwann cell myelination.Furthermore,we observed that 21 days after inducing a sciatic nerve crush injury in these mice,most axons had remyelinated at the injury site in the control nerve,while Ezh2^(fl/fl);Mpz-Cre mice had significantly fewer remyelinated axons compared with their wild-type littermates.This suggests that the absence of Ezh2 in Schwann cells impairs myelin formation and remyelination.In conclusion,EZH2 has emerged as a pivotal regulatory factor in the process of demyelination and myelin regeneration following peripheral nerve injury.Modulating EZH2 activity during these processes may offer a promising therapeutic target for the treatment of peripheral nerve injuries.

Polyethylene glycol has immunoprotective effects on sciatic allografts, but behavioral recovery and graft tolerance require neurorrhaphy and axonal fusion

Behavioral recovery using(viable)peripheral nerve allografts to repair ablation-type(segmental-loss)peripheral nerve injuries is delayed or poor due to slow and inaccurate axonal regeneration.Furthermore,such peripheral nerve allografts undergo immunological rejection by the host immune system.In contrast,peripheral nerve injuries repaired by polyethylene glycol fusion of peripheral nerve allografts exhibit excellent behavioral recovery within weeks,reduced immune responses,and many axons do not undergo Wallerian degeneration.The relative contribution of neurorrhaphy and polyethylene glycol-fusion of axons versus the effects of polyethylene glycol per se was unknown prior to this study.We hypothesized that polyethylene glycol might have some immune-protective effects,but polyethylene glycol-fusion was necessary to prevent Wallerian degeneration and functional/behavioral recovery.We examined how polyethylene glycol solutions per se affect functional and behavioral recovery and peripheral nerve allograft morphological and immunological responses in the absence of polyethylene glycol-induced axonal fusion.Ablation-type sciatic nerve injuries in outbred Sprague–Dawley rats were repaired according to a modified protocol using the same solutions as polyethylene glycol-fused peripheral nerve allografts,but peripheral nerve allografts were loose-sutured(loose-sutured polyethylene glycol)with an intentional gap of 1–2 mm to prevent fusion by polyethylene glycol of peripheral nerve allograft axons with host axons.Similar to negative control peripheral nerve allografts not treated by polyethylene glycol and in contrast to polyethylene glycol-fused peripheral nerve allografts,animals with loose-sutured polyethylene glycol peripheral nerve allografts exhibited Wallerian degeneration for all axons and myelin degeneration by 7 days postoperatively and did not recover sciatic-mediated behavioral functions by 42 days postoperatively.Other morphological signs of rejection,such as collapsed Schwann cell basal lamina tubes,were absent in polyethylene glycol-fused peripheral nerve allografts but commonly observed in negative control and loose-sutured polyethylene glycol peripheral nerve allografts at 21 days postoperatively.Loose-sutured polyethylene glycol peripheral nerve allografts had more pro-inflammatory and less anti-inflammatory macrophages than negative control peripheral nerve allografts.While T cell counts were similarly high in loose-sutured-polyethylene glycol and negative control peripheral nerve allografts,loose-sutured polyethylene glycol peripheral nerve allografts expressed some cytokines/chemokines important for T cell activation at much lower levels at 14 days postoperatively.MHCI expression was elevated in loose-sutured polyethylene glycol peripheral nerve allografts,but MHCII expression was modestly lower compared to negative control at 21 days postoperatively.We conclude that,while polyethylene glycol per se reduces some immune responses of peripheral nerve allografts,successful polyethylene glycol-fusion repair of some axons is necessary to prevent Wallerian degeneration of those axons and immune rejection of peripheral nerve allografts,and produce recovery of sensory/motor functions and voluntary behaviors.Translation of polyethylene glycol-fusion technologies would produce a paradigm shift from the current clinical practice of waiting days to months to repair ablation peripheral nerve injuries.

Silk-based nerve guidance conduits with macroscopic holes modulate the vascularization of regenerating rat sciatic nerve

Peripheral nerve injuries induce a severe motor and sensory deficit. Since the availability of autologous nerve transplants for nerve repair is very limited, alternative treatment strategies are sought, including the use of tubular nerve guidance conduits(tNGCs). However, the use of tNGCs results in poor functional recovery and central necrosis of the regenerating tissue, which limits their application to short nerve lesion defects(typically shorter than 3 cm). Given the importance of vascularization in nerve regeneration, we hypothesized that enabling the growth of blood vessels from the surrounding tissue into the regenerating nerve within the tNGC would help eliminate necrotic processes and lead to improved regeneration. In this study, we reported the application of macroscopic holes into the tubular walls of silk-based tNGCs and compared the various features of these improved silk^(+) tNGCs with the tubes without holes(silk^(–) tNGCs) and autologous nerve transplants in an 8-mm sciatic nerve defect in rats. Using a combination of micro-computed tomography and histological analyses, we were able to prove that the use of silk^(+) tNGCs induced the growth of blood vessels from the adjacent tissue to the intraluminal neovascular formation. A significantly higher number of blood vessels in the silk^(+) group was found compared with autologous nerve transplants and silk^(–), accompanied by improved axon regeneration at the distal coaptation point compared with the silk^(–) tNGCs at 7 weeks postoperatively. In the 15-mm(critical size) sciatic nerve defect model, we again observed a distinct ingrowth of blood vessels through the tubular walls of silk^(+) tNGCs, but without improved functional recovery at 12 weeks postoperatively. Our data proves that macroporous tNGCs increase the vascular supply of regenerating nerves and facilitate improved axonal regeneration in a short-defect model but not in a critical-size defect model. This study suggests that further optimization of the macroscopic holes silk^(+) tNGC approach containing macroscopic holes might result in improved grafting technology suitable for future clinical use.

Concomitant treatment of ureteral calculi and ipsilateral pelvic sciatic nerve schwannoma with transperitoneal laparoscopic approach: A case report

BACKGROUND Schwannomas are rare peripheral neural myelin sheath tumors that originate from Schwann cells.Of the different types of schwannomas,pelvic sciatic nerve schwannoma is extremely rare.Definite preoperative diagnosis of pelvic schwannomas is difficult,and surgical resection is the gold standard for its definite diagnosis and treatment.CASE SUMMARY We present a case of pelvic schwannoma arising from the sciatic nerve that was detected in a 40-year-old man who underwent computed tomography for intermittent right lower back pain caused exclusively by a right ureteral calculus.Subsequently,successful transperitoneal laparoscopic surgery was performed for the intact removal of the stone and en bloc resection of the schwannoma.The total operative time was 125 min,and the estimated blood loss was inconspicuous.The surgical procedure was uneventful.The patient was discharged on postoperative day 5 with the simultaneous removal of the urinary catheter.However,the patient presented with motor and sensory disorders of the right lower limb,caused by partial damage to the right sciatic nerve.No tumor recurrence was observed at the postoperative appointment.CONCLUSION Histopathological examination of the specimen confirmed the diagnosis of a schwannoma.Thus,laparoscopic surgery is safe and feasible for concomitant extirpation of pelvic schwannomas and other pelvic and abdominal diseases that require surgical treatment.

Repair of sciatic nerve defects using tissue engineered nerves

In this study, we constructed tissue-engineered nerves with acellular nerve allografts in Sprague-Dawley rats, which were prepared using chemical detergents-enzymatic digestion and mechanical methods, in combination with bone marrow mesenchymal stem cells of Wistar rats cultured in vitro, to repair 15 mm sciatic bone defects in Wistar rats. At postoperative 12 weeks, electrophysiological detection results showed that the conduction velocity of regenerated nerve after repair with tissue-engineered nerves was similar to that after autologous nerve grafting, and was higher than that after repair with acellular nerve allografts. Immunohistochemical staining revealed that motor endplates with acetylcholinesterase-positive nerve fibers were orderly arranged in the middle and superior parts of the gastrocnemius muscle; regenerated nerve tracts and sprouted branches were connected with motor endplates, as shown by acetylcholinesterase histochemistry combined with silver staining. The wet weight ratio of the tibialis anterior muscle at the affected contralateral hind limb was similar to the sciatic nerve after repair with autologous nerve grafts, and higher than that after repair with acellular nerve allografts. The hind limb motor function at the affected side was significantly improved, indicating that acellular nerve allografts combined with bone marrow mesenchymal stem cell bridging could promote functional recovery of rats with sciatic nerve defects.

Bridging sciatic nerve gap using tissue-engineered nerves constructed with neural tissue-committed stem cells derived from bone marrow

BACKGROUND： Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation. OBJECTIVE： To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells （NTCSCs） derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed in the Anatomical Laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006. MATERIALS： Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawley rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase （NSE）, glial fibrillary acidic protein （GFAP）, and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs. METHODS： All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group： the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group： a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group： a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap. MAIN OUTCOME MEASURES： Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase （HRP, 20%） was injected into the gastrocnemius muscle to retrogradely label the 1-4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method. RESULTS： NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeled neurons in I-45 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group （P 〉 0.05）, but significantly different from the blank nerve scaffold transplantation group （P 〈 0.05）. CONCLUSION＂ NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograft nerve transplantation. ＂

Platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury

The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma.Twenty-eight rabbits were divided into the following groups(7 rabbits/group):model,low-concentrati on PRP(2.5-3.5-fold concentration of whole blood platelets),medium-concentration PRP(4.5-6.5-fold concentration of whole blood platelets),and high-concentration PRP(7.5-8.5-fold concentration of whole blood platelets).Electrophysiological and histomorphometrical assessments and proteomics analysis we re used to evaluate regeneration of the sciatic nerve.Our results showed that platelet-rich plasma containing 4.5-6.5-and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury.Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration.Proteomics analysis showed that after sciatic nerve injury,platelet-rich plasma increased the expression of integrin subunitβ-8(ITGB8),which participates in angiogenesis,and differentially expressed proteins were mainly enriched in focal adhesion pathways.Additionally,two key proteins,ribosomal protein S27 a(RSP27 a)and ubiquilin 1(UBQLN1),which were selected after protein-protein interaction analysis,are involved in the regulation of ubiquitin levels in vivo.These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.

Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair;however,results are still not comparable with the current gold standard technique“autografts”.Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair.Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance.In this study,we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model.Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks.Nevertheless,the molecular assessment in the early regeneration phase(7,14,and 28 days)has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones.Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1,a growth factor that plays an important role in Schwann cell transdifferentiation.The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2,VEGF-A,BDNF,c-Jun,and ATF3.

Long noncoding RNA H19 regulates degeneration and regeneration of injured peripheral nerves

Our previous studies have shown that long noncoding RNA(lncRNA)H19 is upregulated in injured rat sciatic nerve during the process of Wallerian degeneration,and that it promotes the migration of Schwann cells and slows down the growth of dorsal root ganglion axons.However,the mechanism by which lncRNA H19 regulates neural repair and regeneration after peripheral nerve injury remains unclear.In this study,we established a Sprague-Dawley rat model of sciatic nerve transection injury.We performed in situ hybridization and found that at 4–7 days after sciatic nerve injury,lncRNA H19 was highly expressed.At 14 days before injury,adeno-associated virus was intrathecally injected into the L4–L5 foramina to disrupt or overexpress lncRNA H19.After overexpression of lncRNA H19,the growth of newly formed axons from the sciatic nerve was inhibited,whereas myelination was enhanced.Then,we performed gait analysis and thermal pain analysis to evaluate rat behavior.We found that lncRNA H19 overexpression delayed the recovery of rat behavior function,whereas interfering with lncRNA H19 expression improved functional recovery.Finally,we examined the expression of lncRNA H19 downstream target SEMA6D,and found that after lncRNA H19 overexpression,the SEMA6D protein level was increased.These findings suggest that lncRNA H19 regulates peripheral nerve degeneration and regeneration through activating SEMA6D in injured nerves.This provides a new clue to understand the role of lncRNA H19 in peripheral nerve degeneration and regeneration.

Charcot-Marie-Tooth-1A and sciatic nerve crush rat models:insights from proteomics

The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlight and compare the protein signature of these two pathological situations.Indeed,the identification of protein profiles in diseases can play an important role in the development of pharmacological targets.In fact,Charcot-Marie-Tooth-1A rats develop motor impairments that are more severe in the hind limbs.Therefore,for the first time,protein expression in sciatic nerve of Charcot-Marie-Tooth-1A rats was examined.First,distal sciatic nerves were collected from Charcot-Marie-Tooth-1A and uninjured wild-type rats aged 3 months.After protein extraction,sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry was employed.445 proteins mapped to Swiss-Prot or trEMBL Uniprot databases were identified and quantified.Of these,153 proteins showed statistically significant differences between Charcot-Marie-Tooth-1A and wild-type groups.The majority of these proteins were overexpressed in Charcot-Marie-Tooth-1A.Hierarchical clustering and functional enrichment using Gene Ontology were used to group these proteins based on their biological effects concerning Charcot-Marie-Tooth-1A pathophysiology.Second,proteomic characterization of wild-type rats subjected to sciatic nerve crush was performed sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry.One month after injury,distal sciatic nerves were collected and analyzed as described above.Out of 459 identified proteins,92 showed significant differences between sciatic nerve crush and the uninjured wild-type rats used in the first study.The results suggest that young adult Charcot-Marie-Tooth-1A rats(3 months old)develop compensatory mechanisms at the level of redox balance,protein folding,myelination,and axonogenesis.These mechanisms seem insufficient to hurdle the progress of the disease.Notably,response to oxidative stress appears to be a significant feature of Charcot-Marie-Tooth-1A,potentially playing a role in the pathological process.In contrast to the first experiment,the majority of the proteins that differed from wild-type were downregulated in the sciatic nerve crush group.Functional enrichment suggested that neurogenesis,response to axon injury,and oxidative stress were important biological processes.Protein analysis revealed an imperfect repair at this time point after injury and identified several distinguishable proteins.In conclusion,we suggest that peripheral neuropathies,whether of a genetic or traumatic cause,share some common pathological pathways.This study may provide directions for better characterization of these models and/or identifying new specific therapeutic targets.