Tissue engineering for the repair of peripheral nerve injury

Peripheral nerve injury is a common clinical problem and affects the quality of life of patients. Traditional restoration methods are not satisfactory. Researchers increasingly focus on the field of tissue engineering. The three key points in establishing a tissue engineering material are the biological scaffold material, the seed cells and various growth factors. Understanding the type of nerve injury, the construction of scaffold and the process of repair are necessary to solve peripheral nerve injury and promote its regeneration. This review describes the categories of peripheral nerve injury, fundamental research of peripheral nervous tissue engineering and clinical research on peripheral nerve scaffold material, and paves a way for related research and the use of conduits in clinical practice.

Local administration of icariin contributes to peripheral nerve regeneration and functional recovery

Our previous study showed that systemic administration of the traditional Chinese medicine Epimedium extract promotes peripheral nerve regeneration. Here, we sought to explore the ther- apeutic effects of local administration of icariin, a major component of Epimedium extract, on peripheral nerve regeneration. A poly（lactic-co-glycolic acid） biological conduit sleeve was used to bridge a 5 mm right sciatic nerve defect in rats, and physiological saline, nerve growth factor, icariin suspension, or nerve growth factor-releasing microsphere suspension was injected into the defect. Twelve weeks later, sciatic nerve conduction velocity and the number of myelinated fibers were notably greater in the rats treated with icariin suspension or nerve growth factor-releasing microspheres than those that had received nerve growth factor or physiological saline. The effects of icariin suspension were similar to those of nerve growth factor-releasing microspheres. These data suggest that icariin acts as a nerve growth factor-releasing agent, and indicate that local ap- plication of icariin after spinal injury can promote peripheral nerve regeneration.

Biological conduit small gap sleeve bridging method for peripheral nerve injury: regeneration law of nerve fibers in the conduit

The clinical effects of 2-mm small gap sleeve bridging of the biological conduit to repair periph- eral nerve injury are better than in the traditional epineurium suture, so it is possible to replace the epineurium suture in the treatment of peripheral nerve injury. This study sought to identify the regeneration law of nerve fibers in the biological conduit. A nerve regeneration chamber was constructed in models of sciatic nerve injury using 2-mm small gap sleeve bridging of a biodegradable biological conduit. The results showed that the biological conduit had good his- tocompatibility. Tissue and cell apoptosis in the conduit apparently lessened, and regenerating nerve fibers were common. The degeneration regeneration law of Schwann cells and axons in the conduit was quite different from that in traditional epineurium suture. During the prime period for nerve fiber regeneration （2-8 weeks）, the number of Schwann cells and nerve fibers was higher in both proximal and distal ends, and the effects of the small gap sleeve bridging method were better than those of the traditional epineurium suture. The above results provide an objec- tive and reliable theoretical basis for the clinical application of the biological conduit small gap sleeve bridging method to repair peripheral nerve injury.

GSK3β inhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury

Delay of axon regeneration after peripheral nerve injury usually leads to progressive muscle atrophy and poor functional recovery. The Wnt/β-catenin signaling pathway is considered to be one of the main molecular mechanisms that lead to skeletal muscle atrophy in the elderly. We hold the hypothesis that the innervation of target muscle can be promoted by accelerating axon regeneration and decelerating muscle cell degeneration so as to improve functional recovery of skeletal muscle following peripheral nerve injury. This process may be associated with the Wnt/β-catenin signaling pathway. Our study designed in vitro cell models to simulate myelin regeneration and muscle atrophy. We investigated the effects of SB216763, a glycogen synthase kinase 3 beta inhibitor, on the two major murine cell lines RSC96 and C2C12 derived from Schwann cells and muscle satellite cells. The results showed that SB216763 stimulated the Schwann cell migra- tion and myotube contraction. Quantitative polymerase chain reaction results demonstrated that myelin related genes, myelin associated glycoprotein and cyclin-D1, muscle related gene myogenin and endplate-associated gene nicotinic acetylcholine receptors levels were stimulated by SB216763. Immunocytochemical staining revealed that the expressions of ^-catenin in the RSC96 and C2C12 cytosolic and nuclear compartments were increased in the SB216763-treated cells. These findings confirm that the glycogen synthase kinase 3 beta in- hibitor, SB216763, promoted the myelination and myotube differentiation through the Wnt/β-catenin signaling pathway and contributed to nerve remyelination and reduced denervated muscle atrophy after peripheral nerve injury.

Autologous transplantation with fewer fibers repairs large peripheral nerve defects

Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve defects; however, ensuring that the minimum number of transplanted nerve attains maximum therapeutic effect remains poorly understood. In this study, a rat model of common peroneal nerve defect was established by resecting a 10-mm long right common peroneal nerve. Rats receiving transplantation of the common peroneal nerve in situ were designated as the in situ graft group. Ipsilateral sural nerves（10–30 mm long） were resected to establish the one sural nerve graft group, two sural nerves cable-style nerve graft group and three sural nerves cable-style nerve graft group. Each bundle of the peroneal nerve was 10 mm long. To reduce the barrier effect due to invasion by surrounding tissue and connective-tissue overgrowth between neural stumps, small gap sleeve suture was used in both proximal and distal terminals to allow repair of the injured common peroneal nerve. At three months postoperatively, recovery of nerve function and morphology was observed using osmium tetroxide staining and functional detection. The results showed that the number of regenerated nerve fibers, common peroneal nerve function index, motor nerve conduction velocity, recovery of myodynamia, and wet weight ratios of tibialis anterior muscle were not significantly different among the one sural nerve graft group, two sural nerves cable-style nerve graft group, and three sural nerves cable-style nerve graft group. These data suggest that the repair effect achieved using one sural nerve graft with a lower number of nerve fibers is the same as that achieved using the two sural nerves cable-style nerve graft and three sural nerves cable-style nerve graft. This indicates that according to the ‘multiple amplification＇ phenomenon, one small nerve graft can provide a good therapeutic effect for a large peripheral nerve defect.

Temporal changes in the spinal cord transcriptome after peripheral nerve injury

Peripheral nerve injury may trigger changes in mRNA levels in the spinal cord.Finding key mRNAs is important for improving repair after nerve injury.This study aimed to investigate changes in mRNAs in the spinal cord following sciatic nerve injury by transcriptomic analysis.The left sciatic nerve denervation model was established in C57 BL/6 mice.The left L4–6 spinal cord segment was obtained at 0,1,2,4 and 8 weeks after severing the sciatic nerve.mRNA expression profiles were generated by RNA sequencing.The sequencing results of spinal cord mRNA at 1,2,4,and 8 weeks after severing the sciatic nerve were compared with those at 0 weeks by bioinformatic analysis.We identified 1915 differentially expressed mRNAs in the spinal cord,of which 4,1909,and 2 were differentially expressed at 1,4,and 8 weeks after sciatic nerve injury,respectively.Sequencing results indicated that the number of differentially expressed mRNAs in the spinal cord was highest at 4 weeks after sciatic nerve injury.These mRNAs were associated with the cellular response to lipid,ATP metabolism,energy coupled proton transmembrane transport,nuclear transcription factor complex,vacuolar proton-transporting V-type ATPase complex,inner mitochondrial membrane protein complex,tau protein binding,NADH dehydrogenase activity and hydrogen ion transmembrane transporter activity.Of these mRNAs,Sgk1,Neurturin and Gpnmb took part in cell growth and development.Pathway analysis showed that these mRNAs were mainly involved in aldosterone-regulated sodium reabsorption,oxidative phosphorylation and collecting duct acid secretion.Functional assessment indicated that these mRNAs were associated with inflammation and cell morphology development.Our findings show that the number and type of spinal cord mRNAs involved in changes at different time points after peripheral nerve injury were different.The number of differentially expressed mRNAs in the spinal cord was highest at 4 weeks after sciatic nerve injury.These results provide reference data for finding new targets for the treatment of peripheral nerve injury,and for further gene therapy studies of peripheral nerve injury and repair.The study procedures were approved by the Ethics Committee of the Peking University People's Hospital(approval No.2017 PHC004)on March 5,2017.

Use of nerve elongator to repair short-distance peripheral nerve defects: a prospective randomized study

Repair techniques for short-distance peripheral nerve defects, including adjacent joint flexion to reduce the distance between the nerve stump defects, ＂nerve splint＂ suturing, and nerve sle eve connection, have some disadvantages. Therefore, we designed a repair technique involving intraoperative tension-free application of a nerve elongator and obtained good outcomes in the repair of short-distance peripheral nerve defects in a previous animal study. The present study compared the clinical outcomes between the use of this nerve elongator and performance of the conventional method in the repair of short-distance transection injuries in human elbows. The 3-, 6-, and 12-month postoperative follow-up results demonstrated that early neurological function recovery was better in the nerve elongation group than in the conventional group, but no signif- icant difference in long-term neurological function recovery was detected between the two gro ups. In the nerve elongation group, the nerves were sutured without tension, and the duration of postoperative immobilization of the elbow was decreased. Elbow function rehabilitation was significantly better in the nerve elongation group than in the control group. Moreover, there were no security risks. The results of this study confirm that the use of this nerve elongator for repair of short-distance peripheral nerve defects is safe and effective.

Qian-Zheng-San promotes regeneration after sciatic nerve crush injury in rats

Qian-Zheng-San, a traditional Chinese prescription consisting of Typhonii Rhizoma, Bombyx Batryticatus, Scorpio, has been found to play an active therapeutic role in central nervous system diseases. However, it is unclear whether Qian-Zheng-San has therapeutic value for peripheral nerve injury. Therefore, we used Sprague-Dawley rats to investigate this. A sciatic nerve crush injury model was induced by clamping the right sciatic nerve. Subsequently, rats in the treatment group were administered 2 mL Qian-Zheng-San(1.75 g/mL) daily as systemic therapy for 1, 2, 4, or 8 weeks. Rats in the control group were not administered Qian-Zheng-San. Rats in sham group did not undergo surgery and systemic therapy. Footprint analysis was used to assess nerve motor function. Electrophysiological experiments were used to detect nerve conduction function. Immunofluorescence staining was used to assess axon counts and morphological analysis. Immunohistochemical staining was used to observe myelin regeneration of the sciatic nerve and the number of motoneurons in the anterior horn of the spinal cord. At 2 and 4 weeks postoperatively, the sciatic nerve function index, nerve conduction velocity, the number of distant regenerated axons and the axon diameter of the sciatic nerve increased in the Qian-Zheng-San treatment group compared with the control group. At 2 weeks postoperatively, nerve fiber diameter, myelin thickness, and the number of motor neurons in the lumbar spinal cord anterior horn increased in the Qian-Zheng-San treatment group compared with the control group. These results indicate that QianZheng-San has a positive effect on peripheral nerve regeneration.

Sleeve bridging of the rhesus monkey ulnar nerve with muscular branches of the pronator teres: multiple amplification of axonal regeneration

Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previous studies have verified the limit and validity of multiple ampli- fication of peripheral nerve regeneration using small gap sleeve bridging of small donor nerves to repair large receptor nerves in rodents. The present study sought to observe multiple ampli- fication of myelinated nerve fiber regeneration in the primate peripheral nerve. Rhesus monkey models of distal ulnar nerve defects were established and repaired using muscular branches of the right forearm pronator teres. Proximal muscular branches of the pronator teres were su- tured into the distal ulnar nerve using the small gap sleeve bridging method. At 6 months after suture, two-finger flexion and mild wrist flexion were restored in the ulnar-sided injured limbs of rhesus monkey. Neurophysiological examination showed that motor nerve conduction veloc- ity reached 22.63 _＋ 6.34 m/s on the affected side of rhesus monkey. Osmium tetroxide staining demonstrated that the number of myelinated nerve fibers was 1,657 ＋ 652 in the branches of pronator teres of donor, and 2,661 ~ 843 in the repaired ulnar nerve. The rate of multiple amplification of regenerating myelinated nerve fibers was 1.61. These data showed that when muscular branches of the pronator teres were used to repair ulnar nerve in primates, effective regeneration was observed in regenerating nerve fibers, and functions of the injured ulnar nerve were restored to a certain extent. Moreover, multiple amplification was subsequently detected in ulnar nerve axons.

Chitin scaffold combined with autologous small nerve repairs sciatic nerve defects

Although autologous nerve transplantation is the gold standard for treating peripheral nerve defects,it has many clinical limitations.As an alternative,various tissue-engineered nerve grafts have been developed to substitute for autologous nerves.In this study,a novel nerve graft composed of chitin scaffolds and a small autologous nerve was used to repair sciatic nerve defects in rats.The novel nerve graft greatly facilitated regeneration of the sciatic nerve and myelin sheath,reduced atrophy of the target muscle,and effectively restored neurological function.When the epineurium of the small autogenous nerve was removed,the degree of nerve regeneration was similar to that which occurs after autogenous nerve transplantation.These findings suggest that our novel nerve graft might eventually be a new option for the construction of tissue-engineered nerve scaffolds.The study was approved by the Research Ethics Committee of Peking University People's Hospital(approval No.2019 PHE27)on October 18,2019.

Neural regeneration after peripheral nerve injury repair is a system remodelling process of interaction between nerves and terminal effector

In China, there are approximately 20 million people suffering from peripheral nerve injury and this number is increasing at a rate of 2 million per year. These patients cannot live or work independently and are a heavy responsibility on both family and society because of extreme disability and dysfunction caused by peripheral nerve injury （PNI）. Thus, repair of PNI has become a major public health issue in China.

Reinnervation of spinal cord anterior horn cells after median nerve repair using transposition with other nerves

Our previous studies have confirmed that during nerve transposition repair to injured peripheral nerves, the regenerated nerve fibers of motor neurons in the anterior horn of the spinal cord can effectively repair distal nerve and target muscle tissue and restore muscle motor function. To observe the effect of nerve regeneration and motor function recovery after several types of nerve transposition for median nerve defect(2 mm), 30 Sprague-Dawley rats were randomly divided into sham operation group, epineurial neurorrhaphy group, musculocutaneous nerve transposition group, medial pectoral nerve transposition group, and radial nerve muscular branch transposition group. Three months after nerve repair, the wrist flexion test was used to evaluate the recovery of wrist flexion after regeneration of median nerve in the affected limbs of rats. The number of myelinated nerve fibers, the thickness of myelin sheath, the diameter of axons and the cross-sectional area of axons in the proximal and distal segments of the repaired nerves were measured by osmic acid staining. The ratio of newly produced distal myelinated nerve fibers to the number of proximal myelinated nerve fibers was calculated. Wet weights of the flexor digitorum superficialis muscles were measured. Muscle fiber morphology was detected using hematoxylin-eosin staining. The cross-sectional area of muscle fibers was calculated to assess the recovery of muscles. Results showed that wrist flexion function was restored, and the nerve grew into the distal effector in all three nerve transposition groups and the epineurial neurorrhaphy group. There were differences in the number of myelinated nerve fibers in each group. The magnification of proximal to distal nerves was 1.80, 3.00, 2.50, and 3.12 in epineurial neurorrhaphy group, musculocutaneous nerve transposition group, medial pectoral nerve transposition group, and radial nerve muscular branch transposition group, respectively. Nevertheless, axon diameters of new nerve fibers, cross-sectional areas of axons, thicknesses of myelin sheath, wet weights of flexor digitorum superficialis muscle and cross-sectional areas of muscle fibers of all three groups of donor nerves from different anterior horn motor neurons after nerve transposition were similar to those in the epineurial neurorrhaphy group. Our findings indicate that donor nerve translocation from different anterior horn motor neurons can effectively repair the target organs innervated by the median nerve. The corresponding spinal anterior horn motor neurons obtain functional reinnervation and achieve some degree of motor function in the affected limbs.

Repair, protection and regeneration of peripheral nerve injury

Reading guide 1778Repair of long-segment peripheral nerve defects1779Bionic reconstruction of hand function after adult brachial plexus root avulsion1780Optimized design of regeneration material for the treatment of peripheral nerve injury1781Synergism of electroactive polymeric materials and electrical stimulation promotes peripheral nerve repair1783Schwann cell effect on peripheral nerve repair and regeneration .

Large animal models of human cauda equina injury and repair: evaluation of a novel goat model

Previous animal studies of cauda equina injury have primarily used rat models, which display significant differences from humans. Furthermore, most studies have focused on electrophysio- logical examination. To better mimic the outcome after surgical repair of cauda equina injury, a novel animal model was established in the goat. Electrophysiological, histological and magnetic resonance imaging methods were used to evaluate the morphological and functional outcome after cauda equina injury and end-to-end suture. Our results demonstrate successful establish- ment of the goat experimental model of cauda equina injury. This novel model can provide detailed information on the nerve regenerative process following surgical repair of cauda equina injury.

Repair of peripheral nerve defects by nerve transposition using small gap bio-sleeve suture with different inner diameters at both ends

During peripheral nerve transposition repair, if the diameter difference between transposed nerves is large or multiple distal nerves must be repaired at the same time, traditional epineurial neurorrhaphy has the problem of high tension at the suture site, which may even lead to the failure of nerve suture. We investigated whether a small gap bio-sleeve suture with different inner diameters at both ends can be used to repair a 2-mm tibial nerve defect by proximal transposition of the common peroneal nerve in rats and compared the results with the repair seen after epineurial neurorrhaphy. Three months after surgery, neurological function, nerve regeneration, and recovery of nerve innervation muscle were assessed using the tibial nerve function index, neuroelectrophysiological testing, muscle biomechanics and wet weight measurement, osmic acid staining, and hematoxylin-eosin staining. There was no obvious inflammatory reaction and neuroma formation in the tibial nerve after repair by the small gap bio-sleeve suture with different inner diameters at both ends. The conduction velocity, muscle strength, wet muscle weight, cross-sectional area of muscle fibers, and the number of new myelinated nerve fibers in the biosleeve suture group were similar to those in the epineurial neurorrhaphy group. Our findings indicate that small gap bio-sleeve suture with different inner diameters at both ends can achieve surgical suture between nerves of different diameters and promote regeneration and functional recovery of injured peripheral nerves.

Short-term observations of the regenerative potential of injured proximal sensory nerves crossed with distal motor nerves

Motor nerves and sensory nerves conduct signals in different directions and function in different ways.In the surgical treatment of peripheral nerve injuries,the best prognosis is obtained by keeping the motor and sensory nerves separated and repairing the nerves using the suture method.However,the clinical consequences of connections between sensory and motor nerves currently remain unknown.In this study,we analyzed the anatomical structure of the rat femoral nerve,and observed the motor and sensory branches of the femoral nerve in the quadriceps femoris.After ligation of the nerves,the proximal end of the sensory nerve was connected with the distal end of the motor nerve,followed by observation of the changes in the newly-formed regenerated nerve fibers.Acetylcholinesterase staining was used to distinguish between the myelinated and unmyelinated motor and sensory nerves.Denervated muscle and newly formed nerves were compared in terms of morphology,electrophysiology and histochemistry.At 8 weeks after connection,no motor nerve fibers were observed on either side of the nerve conduit and the number of nerve fibers increased at the proximal end.The proportion of newly-formed motor and sensory fibers was different on both sides of the conduit.The area occupied by autonomic nerves in the proximal regenerative nerve was limited,but no distinct myelin sheath was visible in the distal nerve.These results confirm that sensory and motor nerves cannot be effectively connected.Moreover,the change of target organ at the distal end affects the type of nerves at the proximal end.

Comparison of commonly used retrograde tracers in rat spinal motor neurons

The purpose of this study was to investigate the effect of four fluorescent dyes, True Blue（TB）, Fluoro-Gold（FG）, Fluoro-Ruby（FR）, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate（Di I）, in retrograde tracing of rat spinal motor neurons. We transected the muscle branch of the rat femoral nerve and applied each tracer to the proximal stump in single labeling experiments, or combinations of tracers（FG-Di I and TB-Di I） in double labeling experiments. In the single labeling experiments, significantly fewer labeled motor neurons were observed after FR labeling than after TB, FG, or Di I, 3 days after tracer application. By 1 week, there were no significant differences in the number of labeled neurons between the four groups. In the double-labeling experiment, the number of double-labeled neurons in the FG-Di I group was not significantly different from that in the TB-Di I group 1 week after tracer application. Our findings indicate that TB, FG, and Di I have similar labeling efficacies in the retrograde labeling of spinal motor neurons in the rat femoral nerve when used alone. Furthermore, combinations of Di I and TB or FG are similarly effective. Therefore, of the dyes studied, TB, FG and Di I, and combinations of Di I with TB or FG, are the most suitable for retrograde labeling studies of motor neurons in the rat femoral nerve.

Anterior subcutaneous transposition of the ulnar nerve improves neurological function in patients with cubital tunnel syndrome

Although several surgical procedures exist for treating cubital tunnel syndrome, the best surgical option remains controversial. To evaluate the efficacy of anterior subcutaneous transposition of the ulnar nerve in patients with moderate to severe cubital tunnel syndrome and to analyze prognostic factors, we retrospectively reviewed 62 patients（65 elbows） diagnosed with cubital tunnel syndrome who underwent anterior subcutaneous transposition. Preoperatively, the initial severity of the disease was evaluated using the Mc Gowan scale as modified by Goldberg： 18 patients（28%） had grade IIA neuropathy, 20（31%） had grade IIB, and 27（42%） had grade III. Postoperatively, according to the Wilson ＆ Krout criteria, treatment outcomes were excellent in 38 patients（58%）, good in 16（25%）, fair in 7（11%）, and poor in 4（6%）, with an excellent and good rate of 83%. A negative correlation was found between the preoperative Mc Gowan grade and the postoperative Wilson ＆ Krout score. The patients having fair and poor treatment outcomes had more advanced age, lower nerve conduction velocity, and lower action potential amplitude compared with those having excellent and good treatment outcomes. These results suggest that anterior subcutaneous transposition of the ulnar nerve is effective and safe for the treatment of moderate to severe cubital tunnel syndrome, and initial severity, advancing age, and electrophysiological parameters can affect treatment outcome.

Repair of long segmental ulnar nerve defects in rats by several different kinds of nerve transposition

Multiple regeneration of axonal buds has been shown to exist during the repair of peripheral nerve injury, which confirms a certain repair potential of the injured peripheral nerve. Therefore, a systematic nerve transposition repair technique has been proposed to treat severe peripheral nerve injury. During nerve transposition repair, the regenerated nerve fibers of motor neurons in the anterior horn of the spinal cord can effectively grow into the repaired distal nerve and target muscle tissues, which is conducive to the recovery of motor function. The aim of this study was to explore regeneration and nerve functional recovery after repairing a long-segment peripheral nerve defect by transposition of different donor nerves. A long-segment(2 mm) ulnar nerve defect in Sprague-Dawley rats was repaired by transposition of the musculocutaneous nerve, medial pectoral nerve, muscular branches of the radial nerve and anterior interosseous nerve(pronator quadratus muscle branch). In situ repair of the ulnar nerve was considered as a control. Three months later, wrist flexion function, nerve regeneration and innervation muscle recovery in rats were assessed using neuroelectrophysiological testing, osmic acid staining and hematoxylin-eosin staining, respectively. Our findings indicate that repair of a long-segment ulnar nerve defect with different donor nerve transpositions can reinnervate axonal function of motor neurons in the anterior horn of spinal cord and restore the function of affected limbs to a certain extent.

The anatomical,electrophysiological and histological observations of muscle contraction units in rabbits:a new perspective on nerve injury and regeneration

In the conventional view a muscle is composed of intermediate structures before its further division into microscopic muscle fibers.Our experiments in mice have confirmed this intermediate structure is composed of the lamella cluster formed by motor endplates,the innervating nerve branches and the corresponding muscle fibers,which can be viewed as an independent structural and functional unit.In this study,we verified the presence of these muscle construction units in rabbits.The results showed that the muscular branch of the femoral nerve sent out 4–6 nerve branches into the quadriceps and the tibial nerve sent out 4–7 nerve branches into the gastrocnemius.When each nerve branch of the femoral nerve was stimulated from the most lateral to the medial,the contraction of the lateral muscle,intermediate muscle and medial muscle of the quadriceps could be induced by electrically stimulating at least one nerve branch.When stimulating each nerve branch of the tibial nerve from the lateral to the medial,the muscle contraction of the lateral muscle 1,lateral muscle 2,lateral muscle 3 and medial muscle of the gastrocnemius could be induced by electrically stimulating at least one nerve branch.Electrical stimulation of each nerve branch resulted in different electromyographical waves recorded in different muscle subgroups.Hematoxylin-eosin staining showed most of the nerve branches around the neuromuscular junctions consisted of one individual neural tract,a few consisted of two or more neural tracts.The muscles of the lower limb in the rabbit can be subdivided into different muscle subgroups,each innervated by different nerve branches,thereby allowing much more complex muscle activities than traditionally stated.Together,the nerve branches and the innervated muscle subgroups can be viewed as an independent structural and functional unit.This study was approved by the Animal Ethics Committee of Peking University People’s Hospital(approval No.2019 PHE027)on October 20,2019.