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Peripheral nerve defect is a common clinical disease, and the principle for treatment is to restore nerve continuity. At present, autologous nerve transfer is the main method for repairing nerve defects in clinic, but its application is limited by many disadvantages. In recent years, artificial repair materials without sacrificing autologous nerve have been applied and the safety and effectiveness have been preliminarily confirmed. This paper reviews the domestic and foreign literature reports in recent years.
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Objective:To observe the effect of adipose tissue decellularized matrix hydrogel (DAT-gel) on the repair of sciatic nerve defect in rats.Methods:From April, 2019 to April, 2020, aseptic granular adipose tissue was collected from healthy adult women who underwent thigh or abdominal liposuction in the Department of Plastic Surgery, the First Medical Centre of the PLA General Hospital. Decellularisation and enzymatic digestion of adipose tissue were performed to prepare DAT-gel. Scanning electron microscope (SEM) was used to observe the ultrastructure of the hydrogel, and rheology was employed to test the gel dynamics and viscoelasticity of the hydrogel. A rat model of sciatic nerve defect was established and randomly divided into 3 groups: simple chitin catheter group (Chitin group), DAT-gel plus chitin catheter group (DAT-gel group) and autologous nerve reverse connection group (Autograft group) with 10 rats in each group. At the 12th week after surgery, the general view, function and morphology of the regenerated nerve were observed to evaluate the repairing status of the injured nerve. One-way analysis of variance (one-way ANOVA) was used for data analysis. If the difference between the groups was statistically significant, the Turkey method was further used for pairwise comparison. P<0.05 was considered as statistically significant. Results:The results of SEM showed that the DAT-gel had a three-dimensional structure in porous fibre network. The results of rheological test results showed that the complex viscosity of the hydrogel at 4 ℃ and 37 ℃ were 148.91 mPa·s and 801.29 mPa·s, respectively. DAT-gel underwent a sol-gel phase transition when the temperature had been increased. The results showed that DAT-gel had a good temperature-sensitive effect, and its critical point of sol-gel phase transition was similar to the internal temperature of rat. The results of animal experiments showed that the morphology and function of the regenerated nerve in the DAT-gel group were superior to Chitin group at 12 weeks after surgery, according to macroscopic view of the regenerated nerve, electrophysiology of the nerve, the morphology of the new axon and the target muscle, etc.. There was statistically significant between groups ( P<0.05). Conclusion:DAT-gel can significantly promote a repair of sciatic nerve defects in rats.
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BACKGROUND: The repair of peripheral nerve defects by nerve conduit bridging can provide a suitable microenvironment for nerve regeneration. On one hand, it can provide a unique channel for nerve regeneration, prevent the invasion of peripheral connective tissue and the formation of scars. On the other hand, it can maintain endogenous and exogenous neurotrophic factors, growth factors and other stimulants to promote axon growth. OBJECTIVE: To observe the therapeutic effect of chitosan/polyvinyl alcohol catheter injected with brain-derived neurotrophic factor sustained-release microspheres to bridge peripheral nerve defects. METHODS: Chitosan/polyvinyl alcohol nerve conduit was prepared by repeated freeze-thaw technique. The brain-derived neurotrophic factor microspheres were obtained by polymer-alloys combined with oil-oil emulsion/solvent evaporation method. A 15 mm sciatic nerve defect model was made in the right hindlimb of 60 adult male Sprague-Dawley rats. They were selected and randomly divided into four groups (n=15 per group): group A implanted with autogenous sciatic nerve; group B implanted with chitosan/polyvinyl alcohol nerve catheter, injected with normal saline; group C implanted with chitosan/ polyvinyl alcohol nerve catheter, injected with brain-derived neurotrophic factor solution; group D implanted with chitosan/polyvinyl alcohol nerve catheter, injected with brain-derived neurotrophic factor sustained-release microspheres. General observation, histological inspection, and electrophysiological determination were performed at 4 months after the surgery. This study was approved by the Research Ethics Committee of the Second Hospital of Hebei Medical University. RESULTS AND CONCLUSION: (1) Gross anatomy showed that muscle atrophy in group A and group D was lighter than that in the other two groups. The grafts in four groups were all adhered to the peripheral tissues, and the nerve in the autotransplantation segment was strongly adhered to the peripheral tissues. In group D, the regenerated nerve had connected the distal and proximal nerves, and the regenerated nerve filled the conduit. (2) Electrophysiological examination showed that the latency of group D was shorter than that of groups B and C (P 0.05). (3) Histological observation showed that there were regenerated nerve fibers in groups B, C, and D. The diameter, number and thickness of myelin sheath of group D were larger than those of group B and group C (P 0.05). (4) The results showed that the injection of brain-derived neurotrophic factor microspheres into chitosan/PVA catheter had a long-term promoting effect on peripheral nerve regeneration.
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Objective To evaluate the clinical safety of the collagen nerve scaffold with longitudinally oriented microchannels in bridging peripheral nerve defect.Methods Five patients with 8 peripheral nerve defects of 18 to 30 (mean,23.8) mm in length were involved in the pilot study and treated from July,2017 to March,2018,including 6 digital nerves and 2 medial antebrachial cutaneous nerves.The defects were repaired with the collagen nerve scaffold with longitudinally oriented microchannels independently developed.Routine therapy of prophylactic systemic antibiotics but no immunosuppressive drugs was given to all patients post-operatively.All patients were followed-up by regular review in the outpatient department combined with WeChat and telephone.The clinical safety of the nerve scaffold was preliminarily evaluated through observing the condition of the healing of the local wound and the whole body.The blood routine examineation and biochemical test were detected.The statistical analysis of the measurement data was performed by the analysis of variance of repeated measurement data,and the difference was statistically significant when P＜0.05.Results All patients were followed-up for 7 to 15 months (average,10 months).No adverse events such as infection,allergy,damage of liver and kidney function occurred.The operative incisions healed primarily,with no redness,exudation and rupture in the local area.There was no systemic symptoms such as fever,nausea,vomiting,skin itching,etc.The results of blood routine tests and biochemical tests were normal.The data of tests was compared,and the difference was not statistically significant (P＞0.05).Conclusion The preliminary study shows that it is clinically safe to bridge peripheral nerve defects with the collagen nerve scaffold with longitudinally oriented microchannels.
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To investigate the role of the bionic multi-channel nerve conduit by reducing mis-match of regenerated nerve fibers in the rabbit sciatic nerve defect. Methods The experiment was conducted from July, 2017 to February, 2019. A total of 55 New Zealand white rabbits were randomly divided into two groups (First group, n=30 and Second group, n=25).There were 5 subgroups (n=6) in the first group, which were autograft and cus-tom-anatomic nerve conduits (CANC) with different channel (1-CANC, 2-CANC, 3-CANC, 4-CANC) that implanted to repair the rabbit sciatic nerve defect (10 mm). The electrophysiological, triceps muscle wet weight recovery rate, histological study and ankle index analysis were used to evaluate the treatment of each group at 12 and 24 weeks postoperatively. There were 5 subgroups (n=5) in the second group. The simultaneous retrograde tracing method was applied to compare with the number of mismatched nerve fibers at 24 weeks postoperatively. All data were recorded and analyzed by One-way ANOVA method, the Turkey’s method was used to compare the differences between each subgroup. The difference was considered to be statistically significant if P＜0.05. Results The autograft group showed the best recovery in the electrophysiology, histology study and ankle index at 12 and 24 weeks postoperatively (P＜0.05).Histology results showed that the same number of myelinated nerve fibers in all CANC group (P＞0.05), but di-ameters of nerve fiber and myelin thickness were higher in 2-CANC and 3-CANC [(10.67±0.56) μm,(10.65±0.53) μm, respectively] compared with 1-CANC and 4-CANC groups [(8.43±0.63) μm, (9.03±0.55) μm, respectively].The differ-ences were similar in electrophysiological, wet weight recovery rate of triceps muscle, histological study and ankle index analysis.Simultaneous retrograde tracing showed that the autograft group had highest total number of labeled profiles, but no significant difference of the total number of labeled profile was showed among the CANC groups. However, the 1-CANC group[(7.1±2.4) %] showed highest percentage of the FB-NY-neurons than other CANC groups[(2.7±1.9)% in 2-CANC, (2.5±2.3) % in 3-CANC, and (2.2±1.2)% in 4-CANC](P＜0.05). Conclusion The autograft group showed the best results among all groups.Compared with the 1-CANC group, the 2-CANC and 3-CANC group obtained more mature regenerated nerve fibers and with a fewer mismatch rate.Moreover, that did not affect the number of regenerated fibers.
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Objective: To investigate the effect of the sciatic nerve elongation on pain in rats. Methods: Thirty-six adult male Wistar rats of SPF grade, weighing 250-300 g. Eighteen of them were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A), nerve no-elongation group (group B), and nerve ligation group (group C). The model of 10-mm sciatic nerve defect was established in all 3 groups. The sciatic nerve was extended at a speed of 1 mm/d for 14 days in group A. The group B was only installed with external fixation. The nerve stumps were ligated in the group C. At 3, 7, 10, and 14 days after operation, the foot injury was evaluated by the autotomy scoring scale. At 14 days after operation, the dorsal root ganglia (DRG) of L 4-S 1 spinal cord of rats in each group was observed by tumor necrosis factor α (TNF-α) immunohistochemical staining, and the primary antibodies were replaced by pure serum as negative control group. Another 18 rats were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A1), nerve no-elongation group (group B1), positive control group (group C1). In groups A1 and B1, the 10-mm long sciatic nerve defect model was established by the same method as groups A and B, and then fixed with external fixation. Nerve elongation was done or not done without anesthesia at 3 days after operation. In group C1, no modeling was done and 20 μL 2.5% formaldehyde was injected into the toes. After 90 minutes, the dorsal horn of spinal cord of L 4-S 1 segment of rats was cutting for c-Fos immunohistochemical staining and the number of positive cells was counted. Primary antibodies were replaced with pure serum as negative control group. Results: The autotomy scores of rats in groups B and C gradually increased postoperatively, and group A remained stable at 0.25±0.50. The scores of group C were significantly higher than those of group A and group B at each time point postoperatively ( P0.05). Conclusion: Nerve elongation does not cause obvious pain neither during the operation of elongation nor throughout the whole elongation.
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Objective: To review the research progress of graphene and its derivatives in repair of peripheral nerve defect. Methods: The related literature of graphene and its derivatives in repair of peripheral nerve defect in recent years was extensively reviewed. Results: It is confirmed by in vitro and in vivo experiments that graphene and its derivatives can promote cell adhesion, proliferation, differentiation and neurite growth effectively. They have good electrical conductivity, excellent mechanical properties, larger specific surface area, and other advantages when compared with traditional materials. The three-dimensional scaffold can improve the effect of nerve repair. Conclusion: The metabolic pathways and long-term reaction of graphene and its derivatives in the body are unclear. How to regulate their biodegradation and explain the electric coupling reaction mechanism between cells and materials also need to be further explored.
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The repair, regeneration and functional recovery of peripheral nerve defects are the clinical problems needed to be solved urgently.Researches showed that the hollow conduits of bridging nerve defects only had the functions of support and isolation, but the composite conduits could simulate nerve regeneration microenvironment and possess biological activity, which could effectively guide the migration of Schwann cells and the regeneration of axons to achieve satisfactory effects of restoration and functional recovery.With the development of tissue engineering, regenerative medicine, biomaterial and the techniques of cells and molecular biology, the construction of biomimetic and functional nerve conduits to bridge long nerve defects has attracted more and more attention. This review will provide a brief overview of the construction strategy of nerve conduits repairing long nerve defects of peripheral nerves.
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Objective To investigate the protective effect of umbilical cord mesenchymal stem cells (UCMSCs) on traumatic brain injury (TBI) in rats.Methods Thirty healthy Sprague-Dawley rats (10 rats for each group) were randomly divided into normal control group (normal),model group (injection of saline after TBI) and UCMSCs transplantation group (injection of UCMSCs after TBI).The rats in experimental groups were sacrificed on the 10th day after UCMSCs transplantation.The percentage of UCMSCs in brain tissue was detected by flow cytometry.The pathological changes of brain tissue were observed by hematoxylin-eosin (HE) staining method.The expressions of vascular endothelial growth factor (VEGF),glial fibrillary acidic protein (GFAP) and brain-derived neurotrophic factor (BDNF) in brain tissue were measured by immunohistochemistry and immunofluorescence double staining.The neurological deficit was evaluated by neurological deficit degree.Results The percentage of CD90,CD73 and CD105 cells in the UCMSCs transplantation group was significandtly higher than that in the model group (0.4％ vs 0.1％,P＜0.05).The results of HE staining showed that the brain injury of the transplanted group was alleviated compared with the model group (P＜0.05).The VEGF of the brain tissue in injury area in the UCMSCs transplantation group was higher than that in the model group (P＜0.05).The number of GFAP and BDNF positive cells in the UCMSCs transplantation group was higher than that in the model group (P＜0.05),and the neurological deficit score was also higher than that in the model group (P＜0.05).Conclusions UCMSCs transplantation for the treatment of TBI rats can effectively reduce the vascular damage in the injury area and promote nerve recovery.
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OBJECTIVE: Repair of sensorial nerve defect is an important issue on peripheric nerve surgery. The aim of the present study was to determine the effects of sensory-motor nerve bridging on the denervated dermatomal area, in rats with sensory nerve defects, using a neural cell adhesion molecule (NCAM). METHODS: We compared the efficacy of end-to-side (ETS) coaptation of the tibial nerve for sural nerve defect repair, in 32 Sprague-Dawley rats. Rats were assigned to 1 of 4 groups: group A was the sham operated group, group B rats had sural nerves sectioned and buried in neighboring muscles, group C experienced nerve sectioning and end-to-end (ETE) anastomosis, and group D had sural nerves sectioned and ETS anastomosis was performed using atibial nerve bridge. Neurological evaluation included the skin pinch test and histological evaluation was performed by assessing NCAM expression in nerve terminals. RESULTS: Rats in the denervated group yielded negative results for the skin pinch tests, while animals in the surgical intervention groups (group C and D) demonstrated positive results. As predicted, there were no positively stained skin specimens in the denervated group (group B); however, the surgery groups demonstrated significant staining. NCAM expression was also significantly higher in the surgery groups. However, the mean NCAM values were not significantly different between group C and group D. CONCLUSION: Previous research indicates that ETE nerve repair is the gold standard for peripheral nerve defect repair. However, ETS repair is an effective alternative method in cases of sensorial nerve defect when ETE repair is not possible.
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Animals , Rats , Methods , Muscles , Neural Cell Adhesion Molecules , Peripheral Nerves , Rats, Sprague-Dawley , Skin , Sural Nerve , Tibial NerveABSTRACT
OBJECTIVE: To fabricate salidroside/collagen/polycaprolactone (PCL) nerve conduit composite and to investigate the effect of composite nerve conduits for repairing sciatic nerve defect. METHODS: The salidroside microspheres were prepared by W/O/W method, and the sustained release rate of microspheres was detected. The microspheres containing 10, 20, and 40 μg salidroside were mixed with collagen to prepare the nerve conduit core layer by freeze-drying method. The shell layer of collagen/PCL scaffold material was fabricated by electrospinning technology. The genipin cross-linked salidroside/collagen/PCL nerve conduit composite was prepared. The structure of nerve conduit was observed before and after cross-linked by scanning electron microscope. Thirty-eight Wistar rats were used to make the right sciatic nerve defect model of 15 mm in length, and randomly divided into groups A, B, C, D (n=9), and group E (n=2), then defect was repaired with the collagen/PCL conduit in group A, autologous nerve in group E, the 10, 20, and 40 μg/mL salidroside/collagen/PCL conduit in groups B, C, and D, respectively. The survival of rats was observed. The sciatic functional index (SFI) was evaluated at 1, 3, and 6 months after operation. At 6 months, the tissue of defect area was harvested for the general, electrophysiology, histological, and immunohistochemical[S-100 and peripheral myelin protein 0(P0)] staining observations. RESULTS: Salidroside microspheres showed burst release at 3 days, and then it tended to be stable at 13 days and lasted for 16 days, with a cumulative release rate of 76.59%. SEM showed that the disordered fiber of nerve conduit shell layer after crosslinking became conglutination, shrinkage, and density, and had void. The channels of core layer were clearly visible before and after crosslinking. The rats had no infection or death after operation. The SFI of group E was significantly higher than that of groups A, B, C, and D at 1, 3, and 6 months (P0.05); there was no significant difference in SFI among groups A, B, C, and D at 3 months (P>0.05); SFI was significantly higher in group C than groups A, B, and D and in groups A and B than group D (P0.05) at 6 months. In addition, no significant difference was shown among different time points in the other groups (P>0.05) except groups C and E at 1, 3, and 6 months (P0.05). The histological observation showed that the nerve fiber tissue of groups B, C, and E was obviously more than that of groups A and D, and group C was similar to group E in the nerve fiber arrangement, and the core layer material of each group was completely degraded. Immunohistochemical staining showed that S-100 and P0 proteins expressed in all groups; and the expression level of groups B, C, and E was significantly higher than that of groups A and D, and gradually increased (P0.05), and P0 expression level of group A was significantly lower than that of group D (P<0.05). CONCLUSIONS: Salidroside/collagen/PCL nerve conduit can promote sciatic nerve defect repair.
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SUMMARY Peripheral nerve defects are still a major challenge in clinical practice,and the most com-monly used method of treatment for peripheral nerve defects is nerve transplantation,which has certain limitations and shortcomings,so new repair methods and techniques are needed.The peripheral nerve is elongated in limb lengthening surgery without injury,from which we got inspirations and proposed a new method to repair peripheral nerve defects:peripheral nerve elongation.The peripheral nerve could be elongated by a certain percent,but the physiological change and the maximum elongation range were still unknown.This study discussed the endurance,the physiological and pathological change of peripheral nerve elongation in detail,and got a lot of useful data.First,we developed peripheral nerve extender which could match the slow and even extension of peripheral nerve.Then,our animal experiment result confirmed that the peripheral nerve had better endurance for chronic elongation than that of acute elonga-tion and cleared the extensibility of peripheral nerve and the range of repair for peripheral nerve defects. Our result also revealed the histological basis and changed the rule for pathological physiology of peri-pheral nerve elongation:the most important structure foundation of peripheral nerve elongation was Fon-tana band,which was the coiling of nerve fibers under the epineurium,so peripheral nerve could be stretched for 8.5% -10.0% without injury because of the Fontana band.We confirmed that peripheral nerve extending technology could have the same repair effect as traditional nerve transplantation through animal experiments.Finally,we compared the clinical outcomes between nerve elongation and perfor-mance of the conventional method in the repair of short-distance transection injuries in human elbows, and the post-operative follow-up results demonstrated that early neurological function recovery was better in the nerve elongation group than in the conventional group.On the whole,all of these experimental re-sults revealed the physiological phenomenon of peripheral nerve elongation,and described the physiologi-cal change and stretch range in detail.The systematic research results have filled the blank in this field, which is very helpful for clinical limb lengthening surgery,the design of elongation surgery and the evalu-ation of the peripheral nerve stretch injury.Peripheral nerve elongation will become an innovative treat-ment technology in repairing peripheral nerve defects.
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Objective To explore the clinical efficacy of PRGD composite nerve conduit in the treatment of human large-diameter,critical peripheral nerve defect in upper extremity.Methods From December,2011 to August,2014,19 patients with large-diameter,critical peripheral nerve defect in upper extremity were treated with PRGD composite nerve conduit.The patients were followeded-up periodically.The sensory and motor function recovery,high frequency ultrasound,and EMG were employed to assess the efficacy.Results The patients were followed up for an average time of 12-32 months(mean 21.75 ± 6.86 months),sensory and motor function recovered excellent in 7 patients,satisfactory in 7 patients,tolerable in 3 patients and no improvement in 2 patients were obtained according to the peripheral nerve function assessment standard built by British medical research council,the rate excellent and satisfactory results was 73.7％.Conclusion It is clinically promising to use PRGD composite nerve conduit to repair large-diameter,critical peripheral nerve defect in upper extremity,thus laying a foundation for its further application in clinical practice.
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Objective To evaluate the clinical effect of surgical operation for recovering the function of ulnar nerve with large segment defect. Methods Twentycases with the large segment defect of ulnar nerve were retrospectively analyzed from September 1999 to December 2006 in the hospital.All the patients were treated by the operation of nerve end-to-side neurorrhaphy . The broken end of ulnar nerve was anastomosed with the median nerve. And observed the recovery of the sensation and motion function of the little finger, interosseous muscles and claw hand, then compared with before. Results All cases were followed up for 8 to 36 months. The mean was 16 months. The sensation and motion function of the little fingers had better restoration after operation. No incision infection, anchylosis or myatrophy was occurred.Excellent(M4 + S4 +) ,Good (M3S3), moderate (M2S2), and poor effects (M1S1) were achieved respectively in 6, 4,2 and 0 cases based on the scale of XU' s grading standard.The excellent and good rate was 83.3%. Conclusion The nerve end-to-side neurorrhaphy was a effective treatment for the patients with large segment defect of ulnar nerve.
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[Objective] To develop biomimicking chitosan scaffolds with longitudinally oriented micro-channels,and investigate their efficacy in bridging 15 mm sciatic nerve gap in rats.[Methods]Chitosan scaffolds with longitudinally oriented micro-channels were fabricated using unidirectional freezing-dry methods.The chitosan scaffolds were used to bridge 15 mm nerve defect in rats,and their efficacy in bridging nerve gap was evaluated by morphometric analysis,retrograde labeling,electrophysiological studies and behavioral analysis.[Results]The chitosan scaffolds developed in the present study showed longitudinally oriented micro-channels,which resembled the dimensions of basal lamina channels in normal nerves.Implantation of chitosan scaffold achieved axonal regeneration and functional recovery similar to autograft implantation in vivo.[Conclusion]The chitosan scaffolds have inner microstructures which resemble the basal lamina channels in normal nerves.The chitosan scaffold may be used as an alternative to autograft in bridging nerve gaps.
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[Objective]To observe the nerve regeneration and functional recovery in the adult rats.[Method]The sciatic nerves on the left side of 15 rats were exposed and 1.0 cm long segments of the nerves were removed from the mid-thigh level and replaced by rabbit nerve made acellular through chemical extraction.At 4 months after procedure,the nerve regeneration and function recovery were examined with HE staining,NF-160 immunohistochemical staining,electrophysiological tests and sciatic functional index(SFI).[Result]In the rats repaired by acellular nerves,regenerated axons re-entered into the acellular xeno-nerve segments without excessive sign of inflammation following implantation.As stimuling(1.5 mA,0.1 ms,1.0 Hz) to the proximal sciatic nerves,the implanted segment resulted in motor evoked potentials,which were recorded from posterior tibial muscles.SFI showed a partial recovery of locomotion of the limb with sciatic nerve defect.[Conclusion]Sciatic nerves defect can be repaired by chemical acellular xenogeneous nerves in rats.There was partial functional recovery,which revealed the grafting with chemical acellular xenogeneous nerves may be a promising mehod for nerve defect in clinical.
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[Objective]To evaluate the effect of post-operative rehabilitative nursing of patients repaired with acellular nerve allograft.[Method]From April 2003 to April 2006,39 inpatients with peripheral nerve defect were subjected to receive acellular nerve allograft in order to repair nerve defect.The patients were rehabilitated with special nursing after being operated and discharged.Among of them,21 patients were followed up over 6 months,the effect of treatment was analyzed.[Result]Among 21 patients,16 people had excellent and good effect of treatment and the efficient rate was 71.4%.[Conclusion]Post-operative rehabili tative nursing is important and effective for rehabilitation patients of peripheral nerve injuries repaired with acellular nerve allograft.
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We used sheletal muscle of various lengths to repair defect of peripheral nerves in our animal experiments. Both pedicled skeletal muscular graft and free skeletal muscular graft, as nerve grafting conduits, were compared and observed under electron microscope. The finding showed that free skeletal muscular graft used as nerve grafting conduits were more helpful to the regenenation of nerve fibers, compased with pedicled skeletal musular graft, especially to nerve defect in places with abound tendon such as wrist and ankle.
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Twenty-one rabbits were randomly divided into three groups: experimental group-filling vein with two neural segments being 0.3cm long to bridge 4cm defect of common peroneal nerve, control group-bridging vein directly to the 4cm defect of common peroneal nerve, auto-nerve-grafting group-cutting off a segment (4cm long) from common peroneal nerve and grafting it inversely.After 25 weeks, morphological, electrophysiological and histological examinations were undertaken, which revealed that the experimental group was most similar to the auto-nerve-grafting group in recovery of motion of the limbs, action potential of muscle, nerve conductive velocity, and regenerating density of nerve fibers and axons. It was a failure in control group. Satisfactory results were also achieved in 2 patients with defect of ulnar nerve treated by filling vein with two neural segments. It suggests that our method is feasible.