Static Sciatic Index Research Articles

In silico model for automated calculation of functional metrics in animal models of peripheral nerve injury repair

The rat sciatic nerve model is commonly used to test novel therapies for nerve injury repair. The static sciatic index (SSI) is a useful metric for quantifying functional recovery, and involves comparing an operated paw versus a control paw using a weighted ratio between the toe spread and the internal toe spread. To calculate it, rats are placed in a transparent box, photos are taken from underneath and the toe distances measured manually. This is labour intensive and subject to human error due to the challenge of consistently taking photos, identifying digits and making manual measurements. Although several commercial kits have been developed to address this challenge, they have seen little dissemination due to cost. Here we develop a novel algorithm for automatic measurement of SSI metrics based on video data using casted U-Nets. The algorithm consists of three U-Nets, one to segment the hind paws and two for the two pairs of digits which input into the SSI calculation. A training intersection over union error of 60 % and 80 % was achieved for the back paws and for both digit segmentation U-Nets, respectfully. The algorithm was tested against video data from three separate experiments. Compared to manual measurements, the algorithm provides the same profile of recovery for every experiment but with a tighter standard deviation in the SSI measure. Through the open-source release of this algorithm, we aim to provide an inexpensive tool to more reliably quantify functional recovery metrics to the nerve repair research community.

Fibrin Glue Coating Limits Scar Tissue Formation around Peripheral Nerves.

Scar tissue formation presents a significant barrier to peripheral nerve recovery in clinical practice. While different experimental methods have been described, there is no clinically available gold standard for its prevention. This study aims to determine the potential of fibrin glue (FG) to limit scarring around peripheral nerves. Thirty rats were divided into three groups: glutaraldehyde-induced sciatic nerve injury treated with FG (GA + FG), sciatic nerve injury with no treatment (GA), and no sciatic nerve injury (Sham). Neural regeneration was assessed with weekly measurements of the visual static sciatic index as a parameter for sciatic nerve function across a 12-week period. After 12 weeks, qualitative and quantitative histological analysis of scar tissue formation was performed. Furthermore, histomorphometric analysis and wet muscle weight analysis were performed after the postoperative observation period. The GA + FG group showed a faster functional recovery (6 versus 9 weeks) compared to the GA group. The FG-treated group showed significantly lower perineural scar tissue formation and significantly higher fiber density, myelin thickness, axon thickness, and myelinated fiber thickness than the GA group. A significantly higher wet muscle weight ratio of the tibialis anterior muscle was found in the GA + FG group compared to the GA group. Our results suggest that applying FG to injured nerves is a promising scar tissue prevention strategy associated with improved regeneration both at the microscopic and at the functional level. Our results can serve as a platform for innovation in the field of perineural regeneration with immense clinical potential.

Inversely engineered biomimetic flexible network scaffolds for soft tissue regeneration.

Graft-host mechanical mismatch has been a longstanding issue in clinical applications of synthetic scaffolds for soft tissue regeneration. Although numerous efforts have been devoted to resolve this grand challenge, the regenerative performance of existing synthetic scaffolds remains limited by slow tissue growth (comparing to autograft) and mechanical failures. We demonstrate a class of rationally designed flexible network scaffolds that can precisely replicate nonlinear mechanical responses of soft tissues and enhance tissue regeneration via reduced graft-host mechanical mismatch. Such flexible network scaffold includes a tubular network frame containing inversely engineered curved microstructures to produce desired mechanical properties, with an electrospun ultrathin film wrapped around the network to offer a proper microenvironment for cell growth. Using rat models with sciatic nerve defects or Achilles tendon injuries, our network scaffolds show regenerative performances evidently superior to that of clinically approved electrospun conduit scaffolds and achieve similar outcomes to autologous nerve transplantation in prevention of target organ atrophy and recovery of static sciatic index.

Reinnervation of Paralyzed Limb Muscle by Nerve-Muscle-Endplate Grafting Technique.

We have developed a novel reinnervation technique called nerve-muscle-endplate grafting in the native motor zone (NMEG-NMZ). However, it remains unknown whether the NMEG-NMZ is effective for limb reinnervation. To evaluate the efficacy of the NMEG-NMZ in limb muscle reinnervation. Forty-five adult rats were divided into 3 groups: NMEG, end-to-end anastomosis (EEA, technique control), and denervation control (DC). The left tibialis anterior muscle was denervated by resecting its nerve. For NMEG-NMZ, the denervated tibialis anterior was reinnervated by transferring a NMEG pedicle from the lateral gastrocnemius muscle. Three months after surgery, static toe spread analysis was performed for all rats and muscle force was measured for the rats treated with NMEG and EEA. Muscle weight, myofiber morphology, regenerated axons, and reinnervated motor endplates in the treated muscles were also quantified and compared with those in the DC group. NMEG-NMZ technique resulted in better muscle force recovery (79% of the control) compared with EEA (51% of the control, P = .048). Toe spread analysis in NMEG-NMZ reinnervated muscles showed static sciatic index = -16.8, whereas -41.4 in EEA, P < .0001). The average weight of the NMEG-NMZ reinnervated muscles (86%) was greater than those of the EEA treated (71%) and DC (26%) muscles (all P < .0001). The mean count of the regenerated axons in the muscles with NMEG-NMZ was 76% of the control, which was larger than that in the muscles with EEA (46%), P < .0001. NMEG-NMZ technique has unique advantages and is superior to EEA for muscle reinnervation and functional recovery.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model.

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells (ADSPCs) in peripheral nerve recovery. Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs. This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned (2% oxygen for 72 hours) autologous ADSPCs (n = 9). This treatment mode was compared with three others: fibrin conduit seeded with ADSPCs cultivated under normoxic conditions (n = 9); non-cell-carrying conduit (n = 9); and nerve autograft only (n = 9). A 16-week follow-up included functional testing (sciatic functional index and static sciatic index) as well as postmortem muscle mass analyses and morphometric nerve evaluations (histology, g-ratio, axon density, and diameter). At 8 weeks, the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups, indicating faster functional regeneration. Furthermore, histologic evaluation showed significantly increased axon outgrowth/branching, axon density, remyelination, and a reduced relative connective tissue area. Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts. Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

A swim test for functional assessment of rodent peripheral nerve regeneration

• Video motion analysis of functional recovery in a sciatic nerve injury and repair model in rats. • Dynamic analysis of swimming motion in contrast to static sciatic index. • Simple setup, fast testing sessions and lower costs compared to other locomotion systems. • Functional assessment method to study new treatments to improve peripheral nerve regeneration.

Fabrication of a 3D Printed PCL Nerve Guide: In Vitro and In Vivo Testing.

Nerve guides are medical devices designed to guide proximal and distal ends of injured peripheral nerves in order to assist regeneration of the damaged nerves. A 3D-printed polycaprolactone (PCL) nerve guide using an aligned gelatin-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) electrospun mat, seeded with PC12 and Schwann cells (SCs) is produced. During characterization with microCT and SEM porosity (55%), pore sizes (675± 40µm), and fiber diameters (382± 25µm) are determined. Electrospun fibers have degree of alignment of 7°, indicating high potential for guidance. On Day 14, PC12 cells migrated from proximal to distal end of nerve guide when SCs are seeded on the guide. After 28 days, over 95% of PC12 are alive and aligned. PC12 cells express early differentiation marker beta-tubulin 10 times more than late marker NeuN. In a 10mm rat sciatic nerve injury, functional recovery evaluated by using static sciatic index (SSI) is observed in mat-free guides and guides containing mat and SCs. Nerve conduction velocities are also improved in these groups. Histological stainings showed tissue growth around nerve guides with highest new tissue organization being observed with mat and cell-free guides. These suggest 3D-printed PCL nerve guides have significant potential for treatment of peripheral nerve injuries.

Effects of photobiomodulation on different application points and different phases of complex regional pain syndrome type I in the experimental model.

BackgroundComplex regional pain syndrome type I (CRPS-I) consists of disorders caused by spontaneous pain or induced by some stimulus. The objective was to verify the effects of photobiomodulation (PBM) using 830 nm wavelength light at the affected paw and involved spinal cord segments during the warm or acute phase.MethodsFifty-six mice were randomized into seven groups. Group (G) 1 was the placebo group; G2 and G3 were treated with PBM on the paw in the warm and acute phase, respectively; G4 and G5 treated with PBM on involved spinal cord segments in the warm and acute phase, respectively; G6 and G7 treated with PBM on paw and involved spinal cord segments in the warm and acute phase, respectively. Edema degree, thermal and mechanical hyperalgesia, skin temperature, and functional quality of gait (Sciatic Static Index [SSI] and Sciatic Functional Index [SFI]) were evaluated.ResultsEdema was lower in G3 and G7, and these were the only groups to return to baseline values at the end of treatment. For thermal hyperalgesia only G3 and G5 returned to baseline values. Regarding mechanical hyperalgesia, the groups did not show significant differences. Thermography showed increased temperature in all groups on the seventh day. In SSI and SFI assessment, G3 and G7 showed lower values when compared to G1, respectively.ConclusionsPBM irradiation in the acute phase and in the affected paw showed better results in reducing edema, thermal and mechanical hyperalgesia, and in improving gait quality, demonstrating efficacy in treatment of CRPS-I symptoms.

Non-thermal plasma application enhances the recovery of transected sciatic nerves in rats.

This experimental research aimed to investigate the effects of non-thermal plasma on nerve regeneration after transected nerve damage using the sciatic nerve in Wistar albino (A) rats. The experiments were performed on 27 Wistar A rats. The rats underwent surgery for right sciatic nerve exposure and were divided into three groups (each group, n = 9) according to sciatic nerve transected injury (SNTI) and non-thermal plasma application: a non-nerve damage (non-ND) group, a only nerve damage without non-thermal plasma application (ND) group, and a nerve damage with non-thermal plasma application (ND + NTP) group. Subsequent to SNTI and immediate suture, non-thermal plasma was administered three times per week for eight weeks. Evaluation for functional recovery was performed using the static sciatic index measured over the full treatment period of eight weeks. The sciatic nerve specimens were obtained after euthanasia and third day from the last non-thermal plasma application. The sciatic nerve tissues were subjected to histological analysis. Behavior analysis presented that the ND + NTP group showed improved static sciatic index compared with the nerve damage group. Histopathological findings demonstrated that the ND + NTP group had more dense Schwann cells and well-established continuity of nerve fibers, greater than the nerve damage group. Immunohistochemistry showed that the ND + NTP group had increased levels of markers for microtubule-associated protein 2 (MAP2), tau, S100 calcium-binding protein B, and neurofilament-200 and regulated the overexpression of CD68 and MAP2. These results indicated that non-thermal plasma enhanced the motor function and restored the neuronal structure by accelerating myelination and axonal regeneration. Additionally, non-thermal plasma was confirmed to have a positive effect on the recovery of SNTI in rats.

High energy photobiomodulation therapy in the early days of injury improves sciatic nerve regeneration in mice

Introduction: Different studies have evaluated the effects of electrophysical agents on regeneration after peripheral nerve injury. Among them, the most used in clinical and experimental research is photobiomodulation therapy (PBMT). Objective: To analyze the effect of standard energy (16.8 J) of PBMT on peripheral nerve regeneration, applied at different periods after sciatic nerve injury in mice. Methods: Thirty male Swiss mice were divided into six groups: naive; sham; control; LLLT-01 (660 nm, 16.8 J of total energy emitted in 1 day); LLLT-04 (660 nm, 4.2 J per day, 16.8 J of total energy emitted in 4 days); LLLT-28, (660 nm, 0.6 J per day, 16.8 J of total energy emitted over 28 days). The animals were evaluated using thermal hyperalgesia, Sciatic Functional Index (SFI), and Static Sciatic Index (SSI). Data were obtained at baseline and after 7, 14, 21, and 28 days after surgery. Results: For the SFI and SSI, all groups showed significant differences compared to the control group, and the LLLT-04 group presented the best results among those receiving PBMT. In the assessment of thermal hyperalgesia, there was a significant difference in the 14th day of evaluation in the LLLT-04 group. Conclusion: The application of 16.8 J was useful in sciatic nerve regeneration with an improvement of hyperalgesia, with higher efficacy when applied in four days (4.2 J/day).

Mechanical, topographicaland chemical cues combined with physical therapy for peripheral nerve injuries.

Aim: The aim of this paper is to evaluate biomaterial cues combined with physical therapy (PT) on functional recovery in a rat sciatic nerve injury model. Materials & methods: Nerve growth conduits were filled with longitudinally aligned hyaluronic acid fibers and microspheres containing neurotrophic factor (growth factor[GF]). All animals received behavior and functional testing throughout the study, which concluded with measurement of compound muscle action potentials and contractile force of the gastrocnemius muscle. Results & conclusion: Including GF improved recovery of gross motor function and increased sensory pain sensation. During the 4weeks that animals participated in PT, these groups showed higher static sciatic index scores. Including GF and PT has the potential to improve clinical outcomes following peripheral nerve injury.

A novel tissue engineered nerve graft constructed with autologous vein and nerve microtissue repairs a long-segment sciatic nerve defect.

Veins are easy to obtain, have low immunogenicity, and induce a relatively weak inflammatory response. Therefore, veins have the potential to be used as conduits for nerve regeneration. However, because of the presence of venous valves and the great elasticity of the venous wall, the vein is not conducive to nerve regeneration. In this study, a novel tissue engineered nerve graft was constructed by combining normal dissected nerve microtissue with an autologous vein graft for repairing 10-mm peripheral nerve defects in rats. Compared with rats given the vein graft alone, rats given the tissue engineered nerve graft had an improved sciatic static index, and a higher amplitude and shorter latency of compound muscle action potentials. Furthermore, rats implanted with the microtissue graft had a higher density and thickness of myelinated nerve fibers and reduced gastrocnemius muscle atrophy compared with rats implanted with the vein alone. However, the tissue engineered nerve graft had a lower ability to repair the defect than autogenous nerve transplantation. In summary, although the tissue engineered nerve graft constructed with autologous vein and nerve microtissue is not as effective as autologous nerve transplantation for repairing long-segment sciatic nerve defects, it may nonetheless have therapeutic potential for the clinical repair of long sciatic nerve defects. This study was approved by the Experimental Animal Ethics Committee of Chinese PLA General Hospital (approval No. 2016-x9-07) on September 7, 2016.

Effect of Systemic Adipose-derived Stem Cell Therapy on Functional Nerve Regeneration in a Rodent Model.

Lewis rats were divided into 3 groups: 10 mm sciatic nerve resection ("CUT" group; n = 10), transection and repair ("REP" group; n = 10), transection and repair plus single-dose ASCs ("ASC" group; n = 12). Allogeneic (Brown Norway rat) ASCs (1 × 106) were administered intravenously on postoperative day 1. Functional outcome was assessed by static sciatic index, toe spread factor, and a dynamic swim test on a weekly basis for 6 weeks. Sciatic nerves and gastrocnemius muscles were harvested at endpoint (6 weeks) for histological analysis. The ASC group showed accelerated functional recovery on the swim test at 2 weeks postoperatively, with continued improvement over 4 weeks, culminating in superior overall outcomes at 6 weeks compared with the REP group. The CUT group showed no significant improvement from baseline. Nerve histomorphometry correlated well with the swim test results in the ASC group. Gastrocnemius muscle weights showed no difference between the REP and the ASC groups. Our study confirms that early, single dose, systemic administration of ASC after PNI accelerates and enhances overall motor recovery on static and dynamic functional tests as evidenced by improvements in voluntary as well as involuntary motions.

Photobiomodulation in Sciatic Nerve Crush Injuries in Rodents: A Systematic Review of the Literature and Perspectives for Clinical Treatment.

Objective : The aim of the study was to perform a literature review to analyze the effect of photobiomodulation in experimental studies on peripheral nerve regeneration after sciatic nerve crush injury in rodents. Methods: A bibliographic search was performed in the electronic databases, including MEDLINE (PubMed), SCOPUS, and SciELO, from 2008 to 2018. Results: A total of 1912 articles were identified in the search, and only 19 fulfilled all the inclusion criteria. Along with the parameters most found in the manuscripts, the most used wavelengths were 660 nm and 830 nm, power of 30 and 40 mW, and energy density of 4 and 10 J/cm2 . For total energy throughout the intervention period, the lowest energy found with positive effects was 0.70 J, and the highest 1.141 J. Seventeen studies reported positive effects on nerve regeneration. The variables selected to analyze the effect were: Sciatic Functional Index (SFI), Static Sciatic Index (SSI), morphometric, morphological, histological, zymographic, electrophysiological, resistance mechanics and range of motion (ROM). The variety of parameters used in the studies demonstrated that there is yet no pre-determined protocol for treating peripheral nerve regeneration. Only two studies by different authors used the same power, energy density, beam area, and power density. Conclusion: It was concluded that the therapeutic window of the photobiomodulation presents a high variability of parameters with the wavelength (632.8 to 940 nm), power (5 to 170 mW) and energy density (3 to 280 J /cm2 ), promoting nerve regeneration through the expression of cytokines and growth factors that aid in modulating the inflammatory process, improving morphological aspects, restoring the functionality to the animals in a brief period.

Non-thermal plasma accelerates the healing process of peripheral nerve crush injury in rats.

The objective of this study was to evaluate the effect of non-thermal plasma (NTP) on the healing process of peripheral nerve crush injuries, which can occur during dental implant procedures. For this, a rat model of sciatic nerve crush injury (SNCI) was adopted. The rats were divided into three groups: non-nerve damage (non-ND), nerve damage (ND), and ND+NTP group. To evaluate the sciatic nerve (SN) function, the static sciatic index was calculated, and the muscle and SN tissues were subjected to a histologic analysis. The results showed that NTP effectively accelerated the healing process of SNCI in rats. In contrast to the ND group, which showed approximately 60% recovery in the SN function, the NTP-treated rats showed complete recovery. Histologically, the NTP treatments not only accelerated the muscle healing, but also reduced the edema-like phenotype of the damaged SN tissues. In the ND group, the SN tissues had an accumulation of CD68-positive macrophages, partially destroyed axonal fibers and myelinated Schwann cells. Conversely, in the ND+NTP group, the macrophage accumulation was reduced and an overall regeneration of the damaged axon fibers and the myelin sheath was accomplished. The results of this study indicate that NTP can be used for healing of injured peripheral nerves.

Combined delivery of VEGF and IGF-1 promotes functional innervation in mice and improves muscle transplantation in rabbits

Microvascular muscle transfer is the gold standard for reanimation following chronic facial nerve paralysis, however, despite the regenerative capacity of peripheral motor axons, poor reinnervation often results in sub-optimal function. We hypothesized that injection of alginate hydrogels releasing growth factors directly into donor tissue would promote reinnervation, muscle regeneration, and function. A murine model of sciatic nerve ligation and neurorrhaphy was first used to assess the ability of gel delivery of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) to promote functional reinnervation. VEGF + IGF-1 gel delivery to aged mice resulted in prolonged ability to control toe movement, increased toe spreading, and improved static sciatic index score, indicative of improved sciatic nerve and neuromuscular junction function. Further, a 26% increase in muscle fiber area, and 2.8 and 3.0-fold increases in muscle contraction force and velocity, respectively, were found compared to blank alginate in the murine model. This strategy was subsequently tested in a rabbit model of craniofacial gracilis muscle transplantation. Electromyography demonstrated a 71% increase in compound muscle action potential 9 weeks after transplantation following treatment with VEGF + IGF-1 alginate, compared to blank alginate in the rabbit model. Improving functional innervation in transplanted muscle via a hydrogel source of growth factors may enhance the therapeutic outcomes of facial palsy treatments and, more broadly, muscle transplantations.

Novel 3-D helix-flexible nerve guide conduits repair nerve defects

Flexible nerve guide conduits (NGCs) are desired in peripheral nerve reconstruction near the joints. Inspired by the engineered structure of the helical tube, we addressed the problems of conventional NGCs often mentioned in clinical feedback. We developed a type of helix-flexible NGC (HF-NGC) and evaluated its mechanical properties as well as its flexibility, and it performed excellently during in vitro tests. During the in vivo tests, HF-NGCs and conventional nonflexible NGCs (NF-NGCs) were implanted in a rat sciatic nerve defect model. A short-term investigation showed that Schwan cells (SCs) infiltrated into the helical groove, and most of them belonged to the activated SC type. Compared with NF-NGCs, HF-NGCs had fewer apoptotic SCs. In the long term investigation, HF-NGCs maintained flexibility in vivo after 3 months. Analyses of the morphometric parameters of nerve fibers and the static sciatic index showed that the HF-NGCs had similar regeneration outcomes to those of traditional NF-NGCs. Therefore, this style of HF-NGC could be used to repair peripheral nerve damage in a cross-joint region with less tension during operation and easy to postoperative rehabilitation. We believe that the HF-NGC is a potentially valuable candidate for clinical use.

Sericin Nerve Guidance Conduit Delivering Therapeutically Repurposed Clobetasol for Functional and Structural Regeneration of Transected Peripheral Nerves.

Peripheral nerve injury often causes significant function loss. Autologous nerve grafting as a gold-standard repair strategy for treating such an injury is limited by donor nerve supply. Tissue-engineered nerve guidance conduits (TENGCs) as a promising alternative for autografting are challenged by large nerve gaps. Herein, we fabricate a glutaraldehyde-cross-linked sericin nerve guidance conduit (GSC) incorporated with clobetasol, a glucocorticoid receptor agonist, for repairing a 10 mm long sciatic nerve gap in a rat model. The GSC exhibits biocompatibility and regeneration-favorable physicochemical properties. GSC's degradation products promote the secretion of neurotrophic factors in Schwann cells. By repurposing clobetasol for peripheral nerve regeneration, our work uncovers clobetasol's previously unknown functions in promoting Schwann cell proliferation and upregulating the expression of myelin-related genes. Importantly, the implantation of this clobetasol-loaded GSC in vivo leads to successful regeneration of the transected sciatic nerve. Strikingly, the regeneration outcome is functionally comparable to that of autologous nerve grafting (evidenced by three parameters). Specifically, the static sciatic index (SSI), relative reaction time (RRT) and nerve conduction velocity (NCV) in Clobetasol/GSC group are -74.55, 1.30, and 46.4 mm/s at Week 12, respectively, while these parameters are -64.53, 1.23, and 49.8 mm/s in Autograft group. Thus, this work represents the first report unveiling clobetasol's potential in peripheral nerve regeneration, reveals the feasibility of applying a sericin conduit for repairing a large nerve defect, and demonstrates the effectiveness of the clobetasol-loaded-GSC based strategy in transected nerves' regeneration.

Cell activity during peripheral nerve defect repair process using a nerve scaffold.

Peripheral nerve defects, but not artificial nerves, are repaired by endogenous cells. We examined cell activity during the repair process in the presence of autologous nerves and artificial preparations in order to guide future artificial nerve fabrication. PLGA tubes, nerve scaffolds comprising a PLGA tube plus 6,000 fibroin fibers, or autologous nerves were implanted into 10 mm rat sciatic nerve defects (n = 60 per group). Over a period of 1-20 weeks after nerve grafting, sections were stained and imaged to distinguish the cell types present and we quantified the recovery of motor and sensory function in the surgically implanted limb. We observed a decreasing trend in inflammatory cell and fibroblast counts over time which ranked in magnitude as: (PLGA group > nerve scaffold > autologous nerve> sham) and an opposite trend in Schwann cell counts. Differences in withdrawal time from hot water and static sciatic index (SSI) indicated that, after repair, sensory and motor function were best in the sham group, followed by the autologous group, the nerve scaffold group, and the PLGA group. These findings indicate that the inflammatory reaction is significant in the first two weeks after nerve grafting, followed by the rebirth of fibroblasts and Schwann cells, which guide axon regeneration. This inflammatory response was a fundamental stage of peripheral defect repair, but a weaker inflammatory response corresponded to better recovery of sensorimotor functional.

Effects of repeated nerve injuries at different time intervals on functional recovery and nerve innervation

Effects of repeated nerve injuries on functional recovery and nerve innervation were examined in rodents. Crush injuries of the sciatic nerve were inflicted on adult rats and repeated twice or thrice at different time intervals of 1, 2, 3, and 4 weeks. Motor function was assessed by the static sciatic index at 1, 7, 14, 21, 28, 35, 42, 49, and 56 days after the final crush. The rates of nerve innervation of the tibialis anterior muscle, a main muscle innervated by the common peroneal nerve, were evaluated by the quantification of βIII-tubulin-positive nerve terminals and α-bungarotoxin-positive acetylcholine receptors 21 and 56 days after the final crush of triple nerve injuries at 1-, 2-, 3-, and 4-week intervals. Compared with single nerve crush injury, delayed recovery of motor function was observed in repeated crush injuries. In addition, recoveries in the triple crush groups were slower than those in the double crush groups. The rates of reinnervation were lower in the triple crush groups than in the single crush groups, both at 21 days (single: 59.7%; triple: 54.1%–56.1%) and 56 days (single: 88.8%; triple: 72.5%–83.0%) after the final crush, except in the groups with 1-week (triple: 73.8%) and 2-week (triple: 70.5%) intervals at 21 days after the final crush. We concluded that the recovery of motor function was delayed according to the number of repetitions of crush injuries, and that the rates of nerve innervation were still low in the triple crush groups 8 weeks after the final crush.