† These authors contributed equally.
This preliminary research determines whether a
combination of reverse end-to-side neurorrhaphy and rapamycin treatment achieves a better
functional outcome than a single application after prolonged
peripheral nerve injury. We found that the tibial nerve function of the reverse end-to-side + rapamycin group recovered better, with a higher tibial function index value, higher
amplitude recovery rate, and shorter latency delay rate (P
Peripheral nerve injury frequently leads to poor functional recovery, particularly when nerve regeneration is adjourned and/or the injury is far from the target organ [1, 2]. Poor recovery should be attributed to irreversible atrophy of the target muscle [3, 4]. After severe nerve injury, the speed of nerve regeneration is usually limited, leading to nerve denervation of the target muscle for a long time. An effective connection of the nerve with the target muscle has failed skeletal muscle atrophy [5, 6, 7, 8]. Therefore, it is necessary to prevent target muscle atrophy by improving the repair of severe nerve injury.
One solution is the early nerve protection technique for the denervated muscle, namely the traditional end-to-side (ETS) neurorrhaphy technique. This technique consists of transecting the distal stump and then suturing on an adjacent nerve in end-to-side form. Then the ETS neurorrhaphy is transected, and the distal stump is sutured to the proximal stump in an end-to-end fashion after the proximal stump reaches the distal stump [7, 8, 9, 10, 11]. Although these methods can provide innervation for the target muscle before the original axon reaches the distal nerve stump, the stumps undergo Waller degeneration twice before reinnervation by the natural axon, which adversely affects axon regeneration and functional recovery . As a development of this technique, we designed the reverse end-to-side (RETS) neurorrhaphy technique. An epineural window on a distal nerve stump is created in suture with an adjacent nerve in RETS fashion in the RETS procedure. The RETS nerve transfer provides supplementary motor axons to enhance regenerative nerve and supplies early motor endplate reinnervation, protecting the target organs until the original intrinsic axon regeneration has occurs. Previous studies have shown that the RETS technique effectively prevented denervated muscle atrophy and significantly improved the functional outcome in rats [13, 14, 15].
Recent studies showed that the mammalian target of rapamycin (Ra) complex 1 (mTORC1) plays a vital role in protein degradation and leads to muscle atrophy following denervation [16, 17, 18, 19, 20]. After the denervation of skeletal muscle, mTORC1 upregulates the phosphorylation of protein kinase B (PKB)/Akt, which increases the expression of transcription factor forkhead box O (FoxO), muscle-specific E3 ubiquitin ligases atrogin-1 (muscle atrophy F-box protein, MAFbx) and muscle-specific ring finger 1 (MuRF1). It accelerates denervation muscle atrophy [16, 21, 22]. Tang’s group found that the mTORC1-FoxO pathway was inhibited in denervated skeletal muscle by the inhibitor of mTORC1: Ra, which improved the outcomes of denervated gastrocnemius muscle atrophy [23, 24, 25, 26]. These studies suggested that rapamycin could improve the denervated muscle recovery after suffering from peripheral nerve injury.
Irreversible muscle atrophy caused by chronic denervation is the main reason for poor functional recovery following severe nerve injury. We have previously proven that the reverse end-to-side neurorrhaphy technique prevented denervated muscle atrophy and improved the functional outcome [13, 14, 15]. Recent studies indicated the protective effect for denervated muscle atrophy with the treatment of rapamycin . The present study tests the hypothesis that a combination of RETS neurorrhaphy and rapamycin treatment will improve the functional outcome of chronic denervation muscle after peripheral nerve injury has occurred.
Adult female Sprague-Dawley rats (eight weeks, 200–250 g) were used in the present study. Rats were housed under controlled, pathogen-free conditions, 12/12-hour light/dark cycle with free access to pellet food and water.
Rats were randomly grouping into four groups (n = 6 each): the denervation with control cosolvent treatment group (Den group), RETS protection with control cosolvent treatment group (RETS group), denervation with rapamycin treatment group (Den + Ra group) and RETS protection with rapamycin treatment group (RETS + Ra group).
The right hind limb of each animal was operated with an aseptic technique. Briefly, adult rats were anesthetized with sodium pentobarbital (30 mg/kg) by intraperitoneal injection. After removing fur, povidone-iodine was used to disinfect the local skin. A parallel longitudinal incision was made from the hip to the posterior femur, exposing the sciatic nerve from proximal to trifurcation and freeing the tibial, peroneal and sural nerves completely. The denervation was performed as per the following processes: the tibial nerve was cut 6.5 mm away from the proximal part of the tibial nerve into the gastrocnemius muscle. The nerve stumps were covered with a silicone cap  to prevent natural reinnervation (Fig. 1A,C). For the groups of RETS protection in the base of denervation, and epineurium window was set up on the distal stump 3.5 mm away from the position of the tibial nerve entering into the gastrocnemius muscle, 40% of the peroneal nerve on the horizontal level of the transverse of the tibial nerve was separated, and the proximal stump of peroneal nerve was sutured at the epineural window in RETS neurorrhaphy (Fig. 1B,D). Four weeks after the initial surgery, all rats had performed a second surgery by removing the distal and proximal tibial stumps, trimming the scar and suturing two stumps together in an end-to-end (ETE) neurorrhaphy. The surgical wounds of all animals were thoroughly rinsed and closed in layers until the wounds healed.
Schematic diagram of surgical procedures for gastrocnemius muscle (GM) of the right hind limb of rats and intraperitoneal treatment with rapamycin or carrier in rats. Gastrocnemius denervation with rapamycin treatment (A); Gastrocnemius denervation with 40% of peroneal nerve RETS neurorrhaphy protection and rapamycin treatment (B); Gastrocnemius denervation with control cosolvent treatment (C); Gastrocnemius muscle denervation with 40% of peroneal nerve reverse end-to-side neurorrhaphy protection and vehicle treatment (D). TN, tibial nerve; GM, gastrocnemius muscle; PN, peroneal nerve; SN, sural nerve; Stage II, four weeks after Stage I.
The rats were treated with rapamycin (LC Laboratories) or
control cosolvent by intraperitoneal injection after the
first surgery and terminated eight weeks after the second surgery. Rapamycin was
dissolved at 20 mg/mL in ethanol and stored at -20
Tibial Function Index (TFI) analysis was performed after the second operation. Animals were allowed to
walk on a closed footpath (10 cm
The electrophysiological test was performed by computer-aided electromyography (EMG) and a nerve conduction velocity system (NC-STAT; NeuroMetrix, Japan). The rats’ experimental and healthy sides’ tibial nerves were exposed eight weeks after the second surgery. Then electrical stimuli (Intensity 5 V, duration 0.1 ms, frequency 1 Hz) were performed on the tibial nerve stem 3.5 mm proximally away from the tibial nerve. The recording electrodes were placed 2 mm proximally and distally away from the repair point of the tibial nerve to measure the Compound Muscle Action Potentials (CMAP) (Fig. 2). After recording the latency and amplitude, the latency delay rate (LDR) and the amplitude recovery rate (ARR) for each rat were calculated to compare the latency and CMAP of the surgical and contralateral control side.
The electrophysiology tests of the gastrocnemius muscle. The stimulating electrode (Intensity 5 V, duration 0.1 ms, frequency 1 Hz) was placed on the tibial nerve trunk at 3.5 mm proximal of the tibial nerve. The recording electrodes (G 1, G 2) were 2 mm proximally and distally away from the repair point of the tibial nerve. TN, tibial nerve; PN, peroneal nerve; SN, sural nerve; GM, gastrocnemius muscle.
The twitch and tetanic tensions of the gastrocnemius muscle were used to show the recovery of muscle strength. After completing of the electrophysiology, the gastrocnemius muscle was exposed and separated from the surrounding tissues with intact tibial nerve. The hind limbs were constrained with forceps, then the distal tendon of the gastrocnemius muscle was connected to a force sensor through a nylon ligature. 50 Hz electrical stimulation was used for tonic tension. The testing was conducted with a force sensor (JZJ101; Guangzhou Instrument Factory, Guangdong, P.R. China) and a computerized bioelectric signal processing system (SMUP-E; Guangdong General Hospital, Guangdong, P.R. China).
The gastrocnemius muscle with the tibial nerve was freed completely after testing the muscle force. The wet muscle weight was scaled by analytical balance (BSA124S, sartorius). The measurement of the surgical side was compared with the contralateral side and recorded the ratio to show the retention of the wet muscle weight.
After weighing, the gastrocnemius muscle was divided into several parts. One of
the samples was dehydrated through graded ethanol, clarified with xylene,
embedded with paraffin, then sliced at four
Data were analyzed with SPSS 26.0 software (SPSS Inc.). Statistical analysis was
performed using two-tailed Student’s t-tests to compare differences
between two groups. One-way analysis of variance (ANOVA) was used for experiments
that required comparing between three or more groups, including a Tukey’s
post-hoc test. For all experiments, results were considered significant at
There were no systemic or local inflammation or postoperative complications after experiments in rats. No auto-mutilation was found in the experiment. The motor function of each group recovered gradually.
TFI analysis showed that the TFI values of the four groups decreased after
denervation, and gradually increased after the tibial nerve repair (Fig. 3).
The TFI values in the Den + Ra group were significantly higher
than in the Den group at every time point (P
The tibial function index was determined after analysis of the
walking trajectory. The higher the TFI score, the better the functional
recovery. *Den group versus Den + Ra group, #Den group versus RETS group,
The results of the LDR and AAR of the gastrocnemius muscle are displayed in Fig. 4. The AAR in RETS + Ra reached 0.68, significantly higher than those
of the other three groups (P
The LDR in the denervation and Den + Ra groups reached 2.93 and 3.22,
respectively, and these were not significantly different (P = 0.234,
Fig. 4B). However, the LDR in the Den and Den + Ra groups was worse than
the RETS + Ra group and the RETS protection group (1.54, 2.20, P
The LDR and ARR of each rat were determined by calculating surgical side results divided by the contralateral side.
AAR of the gastrocnemius muscle (A); LDR of the gastrocnemius muscle (B). Results
statistically significant at P
The measurements of contractile force included twitch and tetanic tension. The
twitch tension of the gastrocnemius muscle with RETS + Ra protection reached 1.68
Measurement of gastrocnemius contractility. Convulsive tension
(A); tonic tension (B). The ratio of the gastrocnemius muscle mass on the
surgical side to the healthy side (C). Results statistically significant at
The ratio calculated the result of wet muscle weight: surgical
side/contralateral side and the results are shown in Fig. 5C. The wet weight of
the surgical side of the RETS + Ra group was nearly completely retained (0.96),
which was statistically significant compared to the other three groups
The ultrastructure of the gastrocnemius muscle in the four groups is shown in
Fig. 6. It can be observed that the ultrastructure of the gastrocnemius muscle in
the four groups was generally preserved, but all groups showed varying degrees
of fibrous connective tissue hyperplasia. The myofibril cross-sectional area in
the RETS + Ra group was 2234.19
H&E staining cross-section micrographs of gastrocnemius muscle
in four groups. Denervation group (A); Den + Ra group (B); RETS nerve protection
group (C); RETS + Ra group (D); Original magnification
Functional recovery has been disappointing in peripheral nerve injury, particularly when nerve regeneration is adjourned and/or the injury is far from their target organs . Due to the limited speed of nerve regeneration in severe nerve injury, it takes months or even a year for axons to reach the target organ. The denervated skeletal muscle may have atrophied or developed fibrosis, even though the axon has regenerated [2, 6]. An effective connection with the atrophied muscle may have failed, which results in poor functional outcomes in peripheral nerve injury [6, 30, 31]. Therefore, protecting the target muscles from chronic denervation may be an essential strategy in improving the functional outcome after peripheral nerve injury. The present study explored the therapeutic potential of combining RETS and rapamycin treatment by using the model of chronic denervation in the gastrocnemius muscle of rats. We proved that the combination of RETS and rapamycin treatment achieved better functional results concerning the chronic denervation model of rats than the single application. Among the four groups, the tibial nerve function of the RETS + Ra group recovered better, with a higher TFI value, higher ARR, and shorter LDR. Furthermore, RETS + Ra provided better protection for the gastrocnemius muscle, stronger muscle contractility and tetanic tension, and a higher myofibril cross-sectional area.
In the traditional ETS neurorrhaphy technique, an adjacent nerve is partially transected then sutured to the distal stump of the injured nerve in ETE form to provide early protection. However, the distal nerve stump undergoes Waller degeneration twice before reinnervation by the natural axon, affecting axon regeneration and functional recovery . Recently, we developed the traditional ETS neurorrhaphy technique into RETS . The RETS technique allows the injured nerve to re-innervate with the adjacent nerve suture on the perineurial window of the chronically denervated distal nerve stump [14, 15]. Likely, that the anterior interosseous nerve (AIN) transfer techniques in treating cubital tunnel syndrome achieve good results clinically . Previous studies found it was better to choose a mixed nerve as a donor’s nerve because it complements the motor axons required to promote functional recovery of the denervated muscle . It indicated that the ETS neurorrhaphy technique procedure using a donor nerve with a partial neurectomy of 40% was the best choice for effectually treating peripheral nerve injury in rats . Therefore, it is reasonable in this study to separate and suture 40% of the peroneal (mixed) nerve at the epineural window that was set up on the distal stump 3.5 mm away from the position of the tibial nerve entering into the gastrocnemius muscle in the form of RETS neurorrhaphy. The results of TFI, ARR and LDR showed that the recovery of tibial nerve function in the RETS group was significantly higher than in the denervated group, which indicated more rebuild of intact nerve-muscle contacts. The ability of nerves with intact nerve-endplate connection to increase the number of muscle fibers in denervated muscles is 5–8 times greater than that of impaired nerves [34, 35]. It is consistent with the myofibril cross-sectional area and muscle wet weight retention in the RETS group. These partially denervated muscles fully recovered their contractile strength with good protective effects on chronically denervated muscle atrophy.
The essence of denervated muscle atrophy results from more significant muscle protein degradation than synthesis [36, 37, 38]. The mTORC1, one of the effectors that play an essential role in protein regulation, has been identified as being strongly activated after denervation [21, 39, 40]. Tang et al.  showed that mTORC1-FoxO-E3 ubiquitin ligase cascade was activated after denervation, which led to muscle atrophy . By using rapamycin, this “atrophy cascade” was inhibited, and the target muscle was rescued. In this study, the rapamycin treatment group had a larger cross-sectional area and muscle mass retention than the denervation group. The recovery of nerve function in the rapamycin treatment group was better than that of the denervation group. Castets’s finding could explain these results . After skeletal muscle denervation, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogated denervation-induced synaptic remodeling and caused neuromuscular endplate degeneration, which could be reversed by rapamycin.
Of course, there are some limitations to this study. Here, we just chose part of the peroneal nerve as a donor’s nerve. However, whether RETS can be applied to other nerves still needs more investigation. Furthermore, although we have proven that rapamycin was beneficial to prevent denervated skeletal muscle atrophy, the treating time, optimal dose, and frequency and duration of rapamycin treatment are still uncertain, which needs further studies.
In conclusion, our study demonstrates the potential for a combination approach including RETS and rapamycin in treating chronically denervated muscle. The effect of the RETS neurorrhaphy technique combined with rapamycin was significantly better than that of RETS or rapamycin alone. This combination of surgery and drug therapy could provide a new approach to treating peripheral nerve injury.
RETS, reverse end-to-side; Ra, rapamycin; ETS, end-to-side; mTORC1, mammalian target of rapamycin (Ra) complex 1; PKB, protein kinase B; FoxO, factor forkhead box O; MAFbx, muscle atrophy F-box protein; MuRF1, muscle-specific ring finger 1; Den, denervation; ETE, end-to-end; TFI, Tibial Function Index; PL, print length; TE, toe extension; IT, middle toe extension; NPL, normal print length; NTE, normal toe extension; NIT, normal middle toe extension; EPL, experimental print length; ETE, experimental toe extension; EIT, experimental middle toe extension; EMG, electromyography; AIN, anterior interosseous nerve; CMAP, Compound Muscle Action Potentials; LDR, Latency Delay Rate; ARR, Amplitude Recovery Rate; ANOVA, One-way analysis of variance; SD, standard deviation.
QL designed this study. YC and XZ carried out the study and collected the data. YC, WY and BL drafted the manuscript. YM and QZ made critical suggestions for this study. All authors approved the final manuscript.
All work was performed in accordance with the Guiding Opinions on the Treatment of Experimental Animals, Guangdong Province Laboratory Animal Management Regulations and the Laboratory Animal Committee (LAC) of the South China University of Technology Policy on the Humane Care and Use of Vertebrate Animals. Ethics approval was obtained from the Administration Committee of the Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences (Permit Number: 2018537A).
We thank Wenhan Huang for his assistance in manuscript preparation.
This project is supported by the Major Science and Technology Project of Guangdong Province (2015B020225007), Natural Science Foundation of Guangdong Province (2018A0303130206, 2021A1515011008), Guangdong Medical Science and Technology Research Fund (A2019150), and the Program of Science and Technology of Guangzhou (201904010424).
The authors declare no conflict of interest.