All animal-related treatments were carried out in compliance with the Ethical Review Guidelines for Experimental Animals Welfare, approved by the Ethics Committee and Experimental Animal Center of Peking University People’s Hospital in Beijing, China. The study followed the internationally recognized standards outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985). Animal welfare and experimental protocols were strictly maintained throughout the research period. Comprehensive documentation of animal-related data was prepared following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines [12].

The rat Schwann cell line (RSC96) was sourced from the Shanghai Cell Bank of Chinese Academy of Sciences (GNR 6, Shanghai, China). The cell line was validated by PCR and tested negative for mycoplasma. The RSC96 cells exhibit two distinct morphologies: neuron-like and round cells. When the cells reach over-confluence, they tend to adopt a round morphology and become loosely adherent. Additionally, RSC96 cells are negative for surface receptors such as nerve growth factor receptor (NGFR), platelet-derived growth factor receptor alpha (PDGFR$\alpha$), and platelet-derived growth factor receptor beta (PDGFR$\beta$). After resuscitation, the cells were seeded into T25 flask (430639, Corning, NY, USA) and cultured for 24 h at 37 °C in a CO₂ incubator (Steri-Cycle 371, ThermoFisher Scientific, Waltham, MA, USA). Cells density was observed daily by inverted light microscope (ECLIPSE Ts2-FL, Nikon, Tokyo, Japan). When cells reached about 80% confluence, the cells were subcultured via digestion with 0.25% trypsin (25200056, Gibco, Green Island, NY, USA).

The stainless steel mold used for fabricating the conical chitosan conduit is comprised of two cylindrical guiding rods with different diameters, linked together by a conical component. The diameter of thinner guiding rod measures 1.0 mm, whereas the thicker section has a diameter of 1.8 mm. The diameter of the guiding rod plays a critical role in the formation of the nerve conduit, as it directly influences how well it aligns with both the proximal common peroneal nerve and distal common peroneal and tibial nerves.

The conical chitosan conduit fabrication has been described in previous study [4] (Fig. 1). Chitosan powder (C915936, Macklin, Shanghai, China) with a degree of deacetylation greater than 70% was dissolved in 2% acetic acid solution (Beijing Chemical Factory, Beijing, China) to prepare 4% chitosan solution. At the same time, gelatin (G274269, Aladdin, Shanghai, China) was dissolved in warm water to form 1.6% gelatin solution. After mixing the two solutions, bubbles are removed using ultrasonication. The conical mold was immersed in the mixed chitosan solution. The mold surface was uniformly coated with chitosan solution, followed by solidification using 5% NaOH solution (S291866, Aladdin, Shanghai, China). The solidified chitosan was washed with deionized water for three times and dehydrated with acetone (Beijing Chemical Factory) for 2 min, dried at room temperature and placed in deacetylation solution (methanol: acetic anhydride = 1:1) for 10 minutes. Subsequently, the chitosan conduit was carefully detached from the mold to obtain hollow tubes with different inner diameters at both ends. Finally, the chitosan conduit was preserved in 75% ethanol for further animal experiments.

Fig. 1.

Preparation process of conical chitosan conduit. Preparation mold on the left. This Figure was created with Biorender (https://www.biorender.com).

The biocompatibility of conical chitosan conduit was evaluated by live-dead staining and cell counting kit-8 (CCK-8) kits (CK04, Dojindo, Kumamoto, Japan). Briefly, the conical chitosan conduit was immersed in high-sugar complete medium at 37 °C for 72 h. Afterwards, Schwann cells in 24-well plate and 96-well plate were cultured in the extract (conical chitosan conduit soaking liquid) and fresh medium for 24 h and 72 h. Following the protocol provieded with the live-dead staining kit (C2015S, Beyotime, Shanghai, China), Calcein-AM (green fluorescence) and propidium iodide (PI, red fluorescence) were used to stain Schwann cells. The cells were incubated at CO₂ incubator for 30 min, and observed with fluorescence microscope (ECLIPSE Ts2-FL, Nikon, Tokyo, Japan). In addition, the medium was removed from the 96-well plate after 24 h and 72 h, washed once with PBS, and 110 µL mixed medium (Dulbecco’s Modified Eagle’s Medium (DMEM) medium 10:1 CCK-8 reagent) was added to each well. The cells were placed in the incubator for one hour, and the optical density (OD) value of each well was measured by microplate reader (iMark, BioRad, Hercules, CA, USA).

Female Sprague-Dawley (SD) rats (6 weeks, 200–230 g) were sourced from Beijing Vital River Laboratory Animal Technology (Beijing, China). These animals were housed in a controlled environment featuring a 12-hour cycle of light and darkness, with humidity levels maintained at 40%.

A total of 24 animals were randomly divided into four groups (n = 6 per group) (Fig. 2): sham group, chitosan conduit + normal saline group (Chi/NS), chitosan conduit +50 µg/kg MeCbl group (Chi/50 µg/kg), chitosan conduit +200 µg/kg MeCbl group (Chi/200 µg/kg). As previously described [13], rats in each group were anesthetized by inhalation of 2.5% isoflurane (R510-22-10, RWD, Shenzhen, China), and then the skin of the right hind limb of rats was cleaned and disinfected before surgery. After that, the muscle gap was bluntly dissected, and the right common peroneal nerve and tibial nerve were exposed and transected at the 5 mm below the sciatic nerve fork. The proximal common peroneal nerve and distal common peroneal nerve and tibial nerve were bridged with the conical chitosan conduit using nylon suture with small gap of 2 mm. The proximal tibial nerve was sutured on the adjacent muscle in reverse direction to avoid self-repair. In the sham group, the muscle was carefully separated, allowing for the exposure of sciatic nerve and its branches, all while ensuring there was no damage to the nerve. After the surgery, sterile saline solution was administered daily throuth oral gavage to each rat in the sham and Chi/NS groups. In the meantime, each rat in the Chi/50 µg/kg and Chi/200 µg/kg group received oral treatment with methylcobalamin solution (HY-B0586, MedChemExpress LLC, Monmouth Junction, NJ, USA).

Fig. 2.

Schematic illustration of animal model in this study. The sciatic nerve (SN) has two branches, including common peroneal nerve (CPN) and tibial nerve (TN). In the experimental group, the CPN and TN were cut 5 mm away from the point where they split into two branches. A conical chitosan conduit was used to bridge the proximal CPN with the distal CPN and TN. This Figure was created with Biorender (https://www.biorender.com). MeCbl, methylcobalamin.

Rat were fed regularly and their daily progress was carefully recorded after surgery, including food consumption, body movements, recovery of surgical incisions, and movements of surgical limbs. Sixteen weeks post-surgery, an examination was conducted on the exposed common peroneal nerve and tibial nerve to determine if adhesions existed between the nerve structures and the adjacent tissues.

Gait analysis was conducted 16 weeks post-surgery. Gait analysis system (ZS-BT/S, Zhongshi Dichuang Technology, Beijing, China) recorded and evaluated the rats’ footprints. Prior to the experiment, all rats were acclimated to the testing environment. Subsequently, each rat passed through a closed runway consisting of a bottom glass plate and a partition. Footprints from each rat were aptured using a high-speed camera. The sciatic function index (SFI) for each animal was determined using the following formula [13]:

SFI = $\frac{\text{109.5}\left(\text{ETS–NTS}\right)}{\text{NTS}}-\frac{\text{38.3}\left(\text{EPL–NPL}\right)}{\text{NPL}}+\frac{\text{13.3}(\text{EITS–NITS})}{\text{NITS}}$ – 8.8

In this formula, exprimental print length (EPL) denotes the measurement taken from the heel to the apex of the third toe, while normal print length (NPL) indicates the standard length from heel to third toe. Experimental toe spread (ETS) signifies the experimental distance measured from the first to the fifth toe, with normal toe spread (NTS) representing the typical length from the first to the fifth toe. Experimental inter toe spread (EITS) refers to the experimental measurement stretching from the second to the fourth toe, and normal inter toe spread (NITS) is the normal length between the second and fourth toes. An SFI score of 0 indicates normal motor function, and a score of –100 suggests complete impairment of motor function.

Rats were anesthetized by inhaling 2.5% isoflurane (R510-22-10, RWD) at 16 weeks after surgery. We used electrophysiological instruments (Oxford Instruments, Oxford, UK) to record the amplitude and latency of compound motor action potential (CMAP). The stimulating electrode was positioned at distal tibial nerve, and the recording electrodes were inserted into the proximal and distal sides of the gastrocnemius. Then, the amplitude and latency of CMAP in each group were induced by rectangular pulse.

Rats were euthanized throuth carbon dioxide inhalation with a filling rate of 30%–70% per minute, and a segment measuring the distal 5 mm of the tibial nerve conduit was obtained. The distal tibial nerve was fixed using 4% paraformaldehyde (P1110, Solarbio, Beijing, China) and dehydrated in a 30% sucrose solution. Subsequently, the tissue was sliced into transverse sections of 10 µm thickness utilizing a freezing microtome (CM1950 OUVV, Leica, Wetzlar, Germany). In each group, the nerve tissue sections were incubated overnight at 4 °C with primary antibodies NF200 (1:400, N4142, MilliporeSigma, Burlington, MA, USA) and S100 (1:400, S2532, MilliporeSigma). After being washed three times with PBS, the sections underwent incubation for 1 h at room temperature with a mixture of secondary antibodies: Alexa488 (1:500, ab150077, Abcam) and Alexa594 (1:500, ab150116, Abcam, Cambridge, UK). Finally, the sections were sealed with quench-proof tablet containing DAPI (P0131, Beyotime) and observed at 100$\times$ magnification using fluorescence microscope (ECLIPSE Ts2-FL, Nikon).

The regenerated tibial nerve was collected from the distal ends of conical chitosan conduit at 16 weeks post-surgery. The nerve specimens were preserved in 2.5% glutaraldehyde (P1126, Solarbio) for 24 h at 4 °C, followed by staining with 1% osmium tetroxide (251755, Sigma-Aldrich, Taufkirchen, Germany) and dehydrated using gradient concentrations of acetone. The nerve tissue was then embedded in epoxy resin, sliced into semithin transverse sections measuring 700 nm in thichness and ultrathin transverse sections that were 70 nm thick. The semithin sections underwent staining with 1% toluidine blue (G3663, Solarbio) to assess density of myelinated nerve fibers at 200$\times$ magnification, while the ultrathin sections were stained with uranyl acetate and leas citrate to evaluate myelin thickness and diameters of myelinated axons through transmission electron microscopy (TEM, JEM-1400, JEOL, Tokyo, Japan). The images were analyzed with Fiji Image J software (National Institutes of Health, Bethesda, MD, USA).

At 16 weeks post-implantation of a nerve conduit, the right gastrocnemius from the implanted limb and the corresponding muscles from the left limb were harvested and immediately weighed with electronic balance (Sartorius, Beijing, China). The ratio of wet weight (right to left) was calculated. Muscle samples were preserved in GD fixative solution (G1111, Servicebio, Wuhan, Hubei, China) for 48 h at 4 °C, followed by dehydration using 15% and 30% sucrose solution. Subsequently, the gastrocnemius muscle was embedded in paraffin and sliced transversely into sections with a thickness of 10 µm. These sections were then stained using the Masson’s trichrome staining kit (G1340, Solarbio) to visualize muscle fibers. For quantitative analysis, five random fields from each section were chosen, and the cross-sectional area of the muscle fibers was measured using Fiji Image J software.

The results are expressed as the mean $\pm$ SD. Statistical analysis was performed with Graphpad Prism 8.0 (GraphPad Software, CA, USA). Intergroup comparisons were assessed by t-test, while multigroup comparisons were analyzed through one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as p value 0.05.

As shown in Fig. 3, the results of live-dead staining and CCK-8 assay demonstrated that the conical chitosan conduit possessed good biocompatibility. After 24 h and 72 h of treatment with the extracts and complete DMEM medium, the live RSC96 cells were labeled green with Calcein, while PI staining labeled dead cells as red (Fig. 3A). The quantitative analysis of CCK-8 assay further demonstrated that the extracts from conical chitosan conduit had no effect on RSC96 cell viability at 24 h and 72 h compared to DMEM group (p $>$ 0.05, Fig. 3B).

Fig. 3.

Biocompatibility evaluation of conical chitosan conduit. (A) The live rat Schwann cell line (RSC96) was labeled with Calcein (green). The dead RSC96 cells were labeled with propidium iodide (PI, red). Scale bar = 100 µm. (B) Cell counting kit-8 (CCK-8) assay for RSC96 cells treated with extracts and Dulbecco’s Modified Eagle’s Medium (DMEM) at 24 h and 72 h. n = 3 per group. ns, no significance; OD, optical density.

Fig. 4A illustrated that the chitosan conduit is transparent. The inner diameters at each end of the conduit differ, with the narrower end measuring 1.0 mm and the wider end measuring 1.8 mm. A conical structure bridges the two ends. Fig. 4B showed that the diameter of the nerves corresponds to the inner diameter of the conduit. As shown in Fig. 4C, by the 16-week post-surgery mark, the conduit underwent partial absorption, and no noticeable signs of inflammation or neuroma were observed at the nerve suture site.

Fig. 4.

Conical chitosan conduit and nerve transposition repair. (A) Representative image of conical chitosan conduit with different inner diameters. (B) Conical chitosan conduit during transposition repair model surgery. The white arrow indicated the proximal common peroneal nerve. (C) The proximal common peroneal nerve connected the distal common peroneal nerve and tibial nerve at 16 weeks after surgery. The white arrow indicated distal common peroneal nerve (up), and distal tibial nerve (down).

SFI serves as an objective indicator for assessing motor function recovery in rat model. The 3D footprints patterns presented in Fig. 5A demonstrated comparable morphology between left and right footprints in the sham group. Statistical analysis of SFI data (Fig. 5C) revealed significant improvement in both Chi/50 µg/kg and Chi/200 µg/kg groups compared to the Chi/NS group (p 0.01). However, the Chi/200 µg/kg group showed inferior performance relative to the sham group at the 16 weeks (p 0.01).

Fig. 5.

Evaluation of motor function and nerve conduction function. (A) Typical 3D stress distribution patterns for each group at 16 weeks post-surgery. (B) Characteristic electrophysiological recordings of compound muscle action potentials (CMAPs) from different groups. Horizontal axis: ms, Vertical axis: mV. (C) The sciatic nerve function index (SFI) in each gro up. (D) CMAP amplitude measurements for each group. (E) CMAP latency measurements for each group. n = 6 per group. *p 0.05, **p 0.01. Chit, chitosan; Chi/NS, chitosan conduit+normal saline.

Electrophysiological assessments were performed to evaluate the recovery of nerve conductivity on surgical side (Fig. 5B). The CAMP amplitude in the tibial nerve serves as an indicator of the quantity of innervated gastrocnemius fibers, while the CAMP latency correlates with the degree of myelination in regenerated axons. The CAMP amplitude of the right hindlimb in the Chi/NS group was lower compared to the Chi/50 µg/kg and Chi/200 µg/kg group (p 0.05, Fig. 5D), which indicated the methylcobalamin administration improved muscle innervation, and was more effective at high concentration (200 µg/kg). Corresponding to the above results, the CMAP latency was obviously higher in the Chi/NS group without MeCbl treatment (p 0.01, Fig. 5E).

Double NF200/S100 immunofluorescence staining of distal tibial nerve in each group was performed at 16 weeks. As shown in Fig. 6, immunofluorescence analysis demonstrated distinct labeling patterns, with NF200-positive regenerated axons exhibiting green fluorescence and S100-positive Schwann cells displaying red fluorescence. The staining images indicated that more axonal regeneration and myelination in both Chi/50 µg/kg and Chi/200 µg/kg groups compared to the Chi/NS group.

Fig. 6.

Immunoflurescence micrographs illustrating axonal regeneration from different group at 16 weeks post-surgery. Axons are identified by green fluorescence (NF200), while Schwann cells are marked with red fluorescence (S100). The nuclei are highlighted using blue fluorescence (DAPI). Scale bar = 100 µm.

The results of TEM analysis and toluidine blue staining on nerve cross-sections were presented in Fig. 7. Fig. 7A presented toluiding blue-stained images for each group, with myelinated fiber density quantified in Fig. 7D. Quantitative analysis revealed a significant reduction in myelinated fiber density per unit area in the Chi/NS group compared to both Chi/50 µg/kg (p 0.05) and Chi/200 µg/kg (p 0.01) groups. Ultrastructural examination through TEM (Fig. 7B,C) showed enhanced myelin thickness in regenerated tibial nerve fibers from Chi/50 µg/kg and Chi/200 µg/kg groups versus Chi/NS group (p 0.01, Fig. 7E), indicating MeCbl-mediated Schwann cell myelination promotion. Furhtermore, myelinated axon diameters in Chi/NS group were smaller than those in Chi/50 µg/kg (p 0.05) and Chi/200 µg/kg (p 0.01) groups (Fig. 7F).

Fig. 7.

Histological assessment of nerve regeneration. (A) Representative micrograph depicting toluidine-blue staining for each group. Scale bar = 25 µm. (B) Exemplary TEM image corresponding to each group. Scale bar = 2 µm. (C) Enlarged view of the micrograph depicted in (B). Scale bar = 200 nm. (D) Density of myelinated nerve fibers in each group. (E) Thickness measurement of myelinated axons within each group. (F) Determination of myelinated axons diameter across each group. n = 6 per group. *p 0.05, **p 0.01.

Denervated muscles undergo varying degrees of atrophy due to loss of contraction. Gastrocnemius muscles from both hindlimbs were dissected and weighed at 16 weeks post-surgery. Fig. 8A showed typical images of operated and contralateral gastrocnemius muscles in each group. The gastrocnemius wet weight ratio (right/left) was higher in the Chi/50 µg/kg (p 0.05) and Chi/200 µg/kg (p 0.01) groups compared to the Chi/NS group, but lower than the sham group (p 0.01, Fig. 8B). Moreover, the transverse sections of operated gastrocnemius muscle were stained with Masson’s trichrome, and Fig. 8C showed the representative images of Masson’s trichrome staining of gastrocnemius muscle fibers. Using Masson’s trichrome staining, we noticed a notable reduction in the presence of vacant areas between muscle fibers were observed in the sham and Chi/200 µg/kg groups. The muscle fiber cross-sectional area recovery (%) of the Chi/NS group showed a reduction compared to the Chi/50 µg/kg (p 0.05) and Chi/200 µg/kg groups (p 0.01) (Fig. 8D).

Fig. 8.

Histological evaluation of the gastrocnemius. (A) Comparative image showing healthy (left) and injured (right) gastrocnemius muscles. (B) Ratio of wet weights of injured to healthy muscles across groups. (C) Masson’s trichrome staining in transverse sections of the right gastrocnemius. Scale bar = 100 µm. (D) Cross-sectional area recovery index of muscle (%) in each group. n = 6 per group. *p 0.05, **p 0.01.