1 Introduction Peripheral nerve injury is clinically one of the most common neurological diseases with high morbidity. Exogenous nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) can significantly promote Schwann cell proliferation,but systemic application of growth factors has little effect. Furthermore, increasing drug dosage is quite likely to lead to adverse reactions. The topical application of growth factors around the nerve‐damaged areacan result in rapid metabolism of these factors,making the treatment unsustainable^[1]. Thus,small‐gap nerve anastomosis could be a simple nerve repair technique with a positive effect^[2]. In this study,we used the controlled release of growth factors in silicone tubes bridging the small gaps in the nerve stump to provide a more effective option to promote nerve regeneration and repair.

2 Materials and methods 2.1 Reagents and equipment Recombinant rat NGF and bFGF were purchased from PeproTech Co. (Rocky Hill,NJ,USA). NF200 and S100 rabbit antimouse antibody was procured from Sigma Chemical Co. (St. Louis,MO,USA). Fibrin gels were obtained from Shanghai RAAS Blood Products Co.,Ltd. (China). Heparin was used from Tianjin Biochemical Pharmaceutical Co.,Ltd. (China). Electrophysiology instruments and the ASB240U biological signal acquisition system were obtained from Chengdu Instrument Factory (China).

2.2 Animals Twenty‐seven healthy,specific pathogen‐free adult male Sprague Dawley rats (body weight 200-240 g; age 6-8 weeks)were used. The animals were randomly divided into 3 groups (9 in each group): group A (growth factor group),group B (saline group),and group C (suture alone group). Experimental animals were provided by the Experimental Animal Center of Tsinghua University,the operation and animal breeding of which are in line with the relevant provisions of the 1996 edition of the Beijing Laboratory Animal Management Regulations. All experiments were approved by the Local Institutional Ethics Committee.

2.3 Sciatic nerve injury in animal models Rats were anesthetized by intraperitoneal injections of 10% chloral hydrate at adose of 0.35 mL/100 g. After removal of hair from the right hind leg,the 4 limbs of each rat were tied so that the animal was in a fixed prone position on an animal operating table. Iodine swabs were used to prepare the surgical area,which was then covered by sterile gauze. A 2‐cm incision was made on the right side of the limb in the lateral femoral area,and the subcutaneous tissue and fascia layer were cut open to expose the sciatic nerve. Under the stereomicroscope,the sciatic nerve was fully dissected out and transected at a distance of 0.5 cm below the greater sciatic foramen. Under a microscope,the two ends of the nerve were put into the sleeves of a 5‐mm silicone tube (inner diameter 2.5 mm,length 5 mm, wall thickness 0.3 mm). The nerve ends extended into the tube approximately l mm. Using a 9/0 atraumatic needle,the outer membrane of the nerve ends and the wall of the silicone tube were fixed by 2 sutures.

In group A,fibrin gel containing heparin (200 U), NGF (2 μg),and bFGF (2 μg) was injected into the silicone tube; in group B,saline was injected into the silicone tube; and in group C,the nerve transection was sutured only. Low‐dose penicillin was added to the incision to prevent infection. Finally,#4 silk was used to close the wound routinely. All animals were housed separately. After 8 weeks,footprint tests and electrophysiological tests were performed and tissue samples were obtained.

2.4 Sciatic functional index detection The sciatic functional index (SFI) is an important indicator of the degree of recovery of sensory and motor function of the hind limb after sciatic nerve restoration. SFI was measured after 8 weeks with the footprint experiments (walking track analysis)^[3]. A SFI score of 0 indicates normal function,and -100 indicates complete loss of sciatic nerve function.

2.5 Nerve electrophysiological test At 8 weeks after surgery,electrophysiological tests were conducted in all animals under chloral hydrate anesthesia. The sciatic nerve in rats was revealed under a microscope and analyzed using the ASB240U biological signal acquisition system. Two bipolar electrodes were respectively placed at the proximal and distal nerve bridges,maintaining a gap of approximately 1.5 cm. Two recording electrodes were inserted into the belly of the gastrocnemius muscle at a 45° angle. After stimulation of the nerve at the proximal and the distal ends of the bridge (single stimulus of 1 mA current),the action potential waveform was traced. The latency difference and distance between the 2 stimulation points were used to calculate the motor nerve conduction velocity. Nerve conduction velocity is calculated as the distance divided by the time required for impulse conduction.

2.6 Muscle wet weight ratio measurement The muscle wet weight ratio is the ratio of the gastrocnemius wet weight on the operative side to that on the normal side. After electrophysiological testing, the gastrocnemius muscles were removed. After the blood was adsorbed on a filter paper,the muscle wet weight was weighed using an analytical balance and the muscle wet weight ratio was calculated.

2.7 Immunofluorescence staining Eight weeks after nerve anastomosis,the nerve segment frozen sections were cut and stained using NF200 and S100 immunofluorescence. Briefly,after 4% paraformaldehyde fixation,the specimens were immersed in 20% (w/v) sucrose solution until they sank. Then, 10‐μm cross sections were cut with a freezing microtome. At room temperature,the slices were incubated in phosphate‐buffered saline (PBS) containing 1% (w/v) goat serum for 1 h before adding NF200 and S100 antibodies (diluted with 1% PBS at a ratio of 1:500) and stored overnight at 4 °C. The sections were washed in 1% PBS 3 times (5 min each) before adding secondary antibody diluted 1:500 with tetramethylrhodamine isothiocyanate (red,to visualize S100) and fluorescein isothiocyanate (green,to visualize NF200) and stored at room temperature for 1 h. After cleaning,the slides were cover slipped and observed under a fluorescence microscope.

2.8 Semi‐thin sections and transmission electron microscopy Eight weeks after nerve anastomosis,segments of nerve tissue were obtained. After 4% paraformaldehyde fixation for 24 h,they were washed in 1% PBS 3 times (5 min each) and then placed in a 1% (w/v) solution of osmium tetroxide. Specimens were embedded in Epon resin,semi‐thin sections were cut (0.5 μm),and ultrathin sections (80 nm) were obtained. The sectionswere stained with 2% (w/v) toluidine blue and observed under a light microscope. The ultrathin sections were counterstained withlead citrate and uranium acetate and examined under a transmission electron microscope (H‐7650B; Hitachi Limited,Japan).

2.9 Statistical analysis The data are expressed as mean ± standard deviation. Groups were compared by analysis of variance using Prism analysis software (GraphPad Software,San Diego, CA,USA). P values < 0.05 were considered statistically significant.

3 Results 3.1 SFI After 8 weeks,the SFI value in group A was -0.71 ± 7.89,which showed the best recovery. The values in groups B and C were -32.4 ± 8.63 and-36.26 ± 8.31, respectively. However,the differences among the 3 groups were not statistically significant (P > 0.05).

3.2 Electrophysiological test Eight weeks after surgery,the sciatic motor nerve conduction velocity in group A was better (33.89 ± 11.12) than that in group B (31.76 ± 13.16) or group C (15.86 ± 12.37). The differences between groups A and C and between groups B and C were statistically significant (P < 0.05),but the difference between groups A and B was not (P > 0.05).

3.3 Muscle wet weight ratio analysis Eight weeks after surgery,the muscles on the involved sides in all 3 groups were atrophied. The muscle wet weight ratio of group A was the highest (0.45 ± 0.13), followed by group B (0.39 ± 0.16) and then group C (0.35 ± 0.12),but the differences among the 3 groups were not statistically significant (P > 0.05).

3.4 Expression of NF‐200 and S100 Immunofluorescence staining of frozen sciatic nerve sections is shown in Figure 1. Green indicates NF‐200, which is an axonal marker protein,and red indicates S100,a Schwann cell myelin marker protein. The results show that the specimens of each group were wrapped in Schwann cells of myelinated nerve fibers, which were,however,smaller than normal nerve fibers (the diameter of normal nerve fibers reaches approximately 20 μm,whereas the diameter of nascent nerve fibers is 5-15 μm). Compared with groups B and C,nerve regeneration density,and myelin thickness in group A were superior,indicating that NGF gel helps regenerate and repair damaged nerves. In group B,the density was also superior to that of group C,which indicates that NGF gel together with a small gap chamber can significantly improve the quality of nerve regeneration. Meanwhile,a small gap around the regeneration chamber can prevent tissue from invading,provide a good microenvironment for nerve regeneration,and avoid the dislocation and derangement of nerve regeneration caused by direct suturing.

3.5 Newborn myelin detection After 8 weeks,toluidine blue staining showed that in all groups,the myelinated nerve fibers were seen,but the nascent nerve fiber diameter of each group was thinner compared with the normal nerve (Figure 2d), along with the thinner myelin. The diameter of the nascent axons of nerve fibers and the myelin thickness in group A (Figure 2a),group B (Figure 2b),and group C (Figure 2c) were lower than those of a normal nerve (Figure 2d),with no significant differences among the 3 groups. However,the density of the regenerated nerves in groups A (Figure2a) and B (Figure 2b) was greater than that of group C (Figure 2c) in a uniform manner. Quantitative image analysis showed that there were more myelinated nerve fibers in group A than there were in group B or C (Table 1).

3.6 Ultrastructure of nerve regeneration Figure 3 shows the ultrathin transmission electron microscopy sections of sciatic nerve regeneration following nerve damage 8 weeks after surgery and in normal rats. The axon diameter is normally thick and regularly shaped in cross section with high electron density and thick myelination (Figure 3d). In contrast, the nascent axon diameter and myelin thickness in group A (Figure 3a),group B (Figure 3b),and group C (Figure 3c) were decreased compared with normal nerve,and the differences among the 3 groups were not statistically significant (Table 1).

4 Discussion This study shows that small‐gap nerve anastomosis in combination with controlled release of NGF and bFGF appears to promote sciatic nerve regeneration in our rat model. Although among the 3 groups of animals,the differences in SFI,muscle wet weight ratio, nascent axon diameter,and myelin sheath thickness were not statistically significant,the initial data demonstrated that group A had a somewhat better SFI, nerve conduction velocity,muscle wet weight ratio, and regenerated nerve fiber myelin density than those in groups B and C. However,further studies are needed to clarify these points.

The mechanism of achieving a small‐gap nerve anastomosis is superior to the traditional neural endto‐ end anastomosis according to the nerve chemotaxis theory^[4]. Chemotaxis includes nerve tissue specificity, i.e.,nerve growth selectively from the proximal to the distal end,and nerve bundle specificity. Thus,the selective nerve bundle growth from the proximal to the distal nerve bundles and peripheral organ specificity,that is,the selective proximal nerve motor branch,may support growth to the distal nerve,and similar sensory nerve regrowth from the proximal to the distal sensory nerve branch could occur selectively.

For successful regeneration after nerve damage, one of the key issues is to provide a favorable microenvironment for peripheral nerve injury to promote regeneration and functional recovery. The ideal microenvironment of nerve regeneration includes a good blood supply and a chamber that can provide neurotrophic and growth factor regenerative agents^[5]. Lundborg and colleagues^[6] found that after nerve injury,the liquid secreted by the stump contains some growth and neurotrophic factors,which promote axonal and nerve regeneration. However,the chamber can limit the diffusion of active factors needed to maintain the endogenous NGF concentration for nerve regeneration to create a good microenvironment. A nerve gap also allows a certain degree of deviation of the position, e.g.,to reduce the chance of growth or neuroma formation caused by the tightly stitched nerve segments^[7]. Thus,the small‐gap bridging method can accelerate the regeneration of peripheral nerves and improve recovery quality.

Use of exogenous NGF and bFGF can further promote Schwann cell proliferation. The silicone tube regenerative microenvironment provides a reliable tool for the study and application of NGF and bFGF to promote nerve regeneration.

Owing to its excellent biocompatibility,biodegradability, and nontoxicity^[8] adhesive fibrin has been widely used as a hemostatic and controlled drug release material. Fibrin glue can be used as a carrier of NGF and bFGF^{[9, 10]}. However,NGF is easily degraded by proteolytic enzymes,whereas heparin,an anticoagulant produced by mast cells,may combine with NGF and bFGF and likely prevent the loss of NGF and bFGF activity^{[11, 12]}. Given the above properties of heparin,we expect that it can increase the stability of NGF in vivo and provide a uniform steady release^[12]. The role of heparin as a storage vehicle could enhance the bioavailability of NGF and bFGF.

In conclusion, small‐gap anastomosis created a favorable environment for nerve regeneration chemotaxis, which is conducive to releasing factors in such a way as to continue to induce nerve regeneration. At the same time,direct suturing may cause nerve stump distortion,forced displacement,and other renewable phenomena unsuitable for nerve regeneration. Collectively, the combined strategy of growth factor gel with small‐gap nerve anastomosis appears to play a superior role in peripheral nerve repair.

Acknowledgments This work was supported by the National High Technology Research and Development Program of China (863 Program,No. 2012AA020905),and the Chow Tai Fook Medical Research Special Fund (No. 202836019‐03).

Conflict of interests All contributing authors have no conflict of interests.