Improving Nerve and Muscle Function: An Exploration of Targeted Nerve Function Replacement Following Differential Delay Periods in a Rat Model

Improving Nerve and Muscle Function: An Exploration of Targeted Nerve Function Replacement Following Differential Delay Periods in a Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improving Nerve and Muscle Function: An Exploration of Targeted Nerve Function Replacement Following Differential Delay Periods in a Rat Model Chunxiao Tang, Yuanheng Li, Xinxian Fan, Jiamei Guo, Yifeng Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5460332/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Journal of NeuroEngineering and Rehabilitation → Version 1 posted 8 You are reading this latest preprint version Abstract Background Targeted Muscle Reinnervation (TMR) improves real-time control of EMG-based prostheses by connecting severed nerves to adjacent muscles, creating new EMG signals. However, TMR requires cutting original nerve connections, which can cause denervation atrophy and limit functional recovery. As an alternative, Targeted Nerve Function Replacement (TNFR) offers promising potential for limb function restoration. This study evaluates TNFR efficacy in restoring denervated muscle function across different postoperative intervals in a rat model. Methods Thirty Sprague-Dawley rats (220–250 g) were divided into five equal groups (n = 6 per group): control (no transection), denervation (transection without repair), immediate TNFR after median nerve transection, 2-week delayed TNFR, and 4-week delayed TNFR. The median nerve was selected for reinnervation with the musculocutaneous nerve innervating the brachialis muscle serving as the anastomosis target. All assessments were conducted 4 weeks post-TNFR intervention, including intramuscular bipolar EMG recordings (1024 Hz sampling rate), behavioral assessment, muscle tension measurement, dorsal root ganglia (DRG) histology, and spinal cord motor neuron evaluation. Results Our findings revealed that immediate TNFR significantly outperformed delayed interventions across all measured parameters. EMG amplitude and root mean square values were significantly higher in the immediate TNFR group compared to delayed intervention groups (P < 0.05). Similarly, maximum contraction force and maximum tetanic contraction force of the biceps brachii demonstrated significantly superior recovery in the immediate TNFR group versus delayed groups (P < 0.05). Histological examination confirmed significantly greater preservation of sensory neurons in the DRG following immediate TNFR compared to delayed interventions (P < 0.05). Immunofluorescence analysis showed that immediate TNFR better preserved synaptic protein expression (synaptophysin/SYN) in motor neurons of the spinal cord compared to delayed interventions, indicating enhanced preservation of motor neuron function. Notably, immediate TNFR prevented the autophagic behavior observed in rats with delayed or absent intervention, suggesting better neuropathic pain prevention. Conclusion Timing critically influences TNFR outcomes, with immediate intervention yielding optimal restoration of both motor and sensory functions. This study provides valuable insights for optimizing surgical strategies in peripheral nerve injury, with important implications for limb reconstruction, rehabilitation protocols, and prosthetic development. Targeted Nerve Function Replacement (TNFR) Electromyography (EMG) Autophagic Behavior Spinal Cord Dorsal Root Ganglion (DRG) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Maintaining limb functionality is essential for seamless interaction with the environment. However, traumatic injuries, vascular diseases, and neurological disorders can severely impair mobility, leading to chronic pain and psychological distress [ 1 ]. Current commercial prosthetics often suffer from limited functionality and delayed response due to inadequate electromyography (EMG) signal sources, resulting in awkward and inefficient movements. This highlights the urgent need for multifunctional prosthetics with intuitive control, particularly for high-level amputees, which can be achieved through advanced surgical interventions. Kuiken et al. introduced a novel surgical technique known as Targeted Muscle Reinnervation (TMR) [ 2 – 4 ]. This approach involves redirecting nerves from the residual limb to specific muscles, while disconnecting their original neural connections. TMR not only restores motor signals from the residual nerve but also facilitates the collection of EMG signals from the target muscle. This enables regenerated nerve axons to produce action potentials—electrical signals that activate muscles in the residual limb, thereby restoring function [ 5 – 7 ]. Subsequently, limb muscles serve as a pathway for nerve signals to reach the skin surface, generating new EMG signals that can enhance prosthetic control [ 8 ]. TMR technology holds promise for improving EMG prosthetic functionality by potentially rehabilitating joint movements and enabling intricate multi-joint motions [ 9 , 10 ]. Moreover, TMR shows potential in restoring movement capabilities and alleviating discomfort in lower limb amputees, fostering optimism for fully functional prosthetic limbs in the future [ 10 – 12 ]. Compared to the well-established TMR technique, Targeted Nerve Function Replacement (TNFR) offers a novel approach by directly reconnecting the target nerve to its original neural pathway, which may lead to superior outcomes in preserving motor and sensory functions while minimizing denervation-induced atrophy. However, TMR surgery involves severing the original neural connections of the target muscle, which can disrupt the normal activity of proteins and metabolic enzymes within those muscles. Additionally, the severed nerve endings undergo Wallerian degeneration, gradually breaking down over time due to a lack of stimulation from the neuron cell body. This degeneration impairs the ability of regenerated axons or nerve fibers to effectively communicate with distant denervated skeletal muscles, exacerbating muscle weakness and atrophy. During this phase, skeletal muscle volume decreases, leading to a significant reduction or complete loss of contraction function. Muscle fibers may develop severe fibrosis and risk irreversible atrophy, ultimately resulting in the loss of motor and sensory functions [ 11 , 13 ]. To address these challenges, researchers have developed several innovative techniques for nerve repair and reinnervation. It is important to distinguish between three key approaches that have emerged in this field: TMR, Regenerative Peripheral Nerve Interfaces (RPNI), and TNFR. TMR, as previously described, redirects severed nerves from the amputated limb to new target muscles after deliberately denervating those muscles. While effective for prosthetic control, this approach sacrifices the original innervation of the target muscles, potentially leading to complications associated with denervation. RPNI employs a different strategy, utilizing free muscle grafts that are carefully wrapped around the terminal ends of transected peripheral nerves [ 14 , 15 ]. These grafts function as biological amplifiers for nerve signals, providing stable interfaces between peripheral nerves and artificial limbs without necessitating the denervation of existing functional muscles. TNFR, the central focus of this study, represents a fundamentally distinct approach. Rather than redirecting nerves to new muscles (as in TMR) or using muscle grafts (as in RPNI), TNFR establishes a direct end-to-end anastomosis between an intact donor nerve and the original nerve of a target muscle. This sophisticated technique preserves existing neural pathways while providing supplementary neural input to the target muscle. In our precisely engineered TNFR model, we perform a meticulous microsurgical procedure where the median nerve serves as the donor nerve and connects to the musculocutaneous nerve that innervates the brachialis muscle. The anastomosis is strategically positioned at the nerve entry point to the target muscle, as illustrated in Fig. 1 . This critical positioning protects motor neurons from prolonged denervation by maintaining neural connections to the target muscle while redirecting potential damage away from the proximal regions containing vital motor neuron bodies toward more distal nerve segments. The TNFR procedure begins with careful exposure of both the donor nerve (median nerve) and the recipient nerve (musculocutaneous nerve) near its entry point to the brachialis muscle. The recipient nerve undergoes proximal transection, while the donor nerve is partially transected to create a specialized branch for anastomosis, thereby preserving a portion of its original function. This strategic partial transection creates an effective neural pathway that enables signals from the donor nerve to reach the denervated muscle, as depicted in Fig. 3 . TNFR preserves muscle viability and function after significant injury by maintaining some original nerve function while adding new neural input. The timing of TNFR intervention likely affects recovery outcomes, much like the established "golden period" for nerve repair [ 16 , 17 ]. Although this concept is widely discussed, there remains ongoing debate regarding its exact timeframe, suggesting that in clinical practice, the timing of nerve repair may need to be customized based on individual circumstances. Despite recent advances, the specific impact of TNFR surgery timing on functional outcomes remains incompletely understood, necessitating further research to determine optimal intervention windows for maximizing recovery potential. TNFR involves creating an anastomosis between the target nerve and the original nerve of the target muscle, rather than simply implanting the target nerve into the muscle. This method facilitates reinnervation of the target muscle by its original nerve, thereby restoring both motor and sensory functions. Additionally, precise and consistent surgical incisions during nerve repair procedures generally promote faster wound healing and better functional outcomes [ 18 , 19 ]. While studies on related techniques such as RPNI have demonstrated benefits of careful surgical approach [ 15 , 20 ]. TNFR's unique methodology–connecting an intact donor nerve to the recipient nerve near the muscle entry point–warrants specific investigation of its wound healing dynamics. The distinct neural network structure within targeted muscles may enhance reinnervation rates following TNFR. However, various clinical conditions can delay nerve repair interventions, potentially compromising outcomes if the optimal 'golden period' for neural regeneration is missed.This strategic positioning near the muscle entry point helps maintain the nerve's connection to its target muscle, thereby shifting the focus of potential damage to the more distal segments of the nerve rather than the proximal regions containing essential motor neuron structures. This adjustment extends the critical window for effective nerve repair and potentially improves the likelihood of successful reinnervation and functional recovery. This study aimed to comprehensively evaluate the recovery of sensory and motor functions in rats that underwent TNFR surgery at different time points after injury. We established a standardized TNFR model using the median nerve for reinnervation and the musculocutaneous nerve innervating the brachialis muscle for anastomosis. Following surgery, we conducted a multifaceted assessment of recovery through intramuscular EMG signal analysis, precise muscle tension measurements, detailed behavioral assessments, quantitative sensory neuron counts in dorsal root ganglia (DRG), and comprehensive motor neuron evaluation in the spinal cord. 2. Materials and Methods 2.1 Animals and experimental design The selection of Sprague-Dawley rats was based on their extensive use in nerve injury models, which provides a consistent framework for evaluating TNFR interventions. Although this strain is prone to autotomy behaviors post-injury, their high availability and well-characterized physiology make them a preferred model for initial exploratory studies. To address autotomy-related limitations, postoperative care protocols were strictly implemented. Additionally, the experimental design aimed to minimize variability by using age-matched and weight-matched subjects under controlled environmental conditions. Thirty Specific Pathogen-Free (SPF) adult male Sprague-Dawley rats, aged 7–8 weeks with a body weight of 220–250 g, were purchased from the Guangdong Medical Laboratory Animal Center, Guangzhou, China (license No. SCXK (Yue) 2013-0002). The rats were kept in an SPF environment at the Zhuhai campus of Zunyi Medical University, Zhuhai, China.The rats were housed in cages (n = 3/cage) under controlled conditions, with a temperature of 22–26 ℃, relative humidity of 40–60%, and a 12:12 h light-dark cycle, with free access to food and water. The rats were randomly divided into five groups: control, denervated, TNFR, 2-week delayed TNFR (TNFR-2W), and 4-week delayed TNFR (TNFR-4W), with six rats in each group. The study protocol was approved by the Animal Ethics Committee of the Zhuhai campus of Zunyi Medical University, Zhuhai, China (approval No. 2019-2-273) on March 11, 2019. The study was conducted in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) [ 21 ]. 2.1.1 Experimental groups and timeline The experiment was designed to evaluate the effectiveness of TNFR at different time points following denervation. Figure 1 illustrates the experimental timeline for all groups. After acclimatization, all experimental groups except the Control underwent median nerve transection on Day 0. The TNFR group received the TNFR procedure immediately after nerve transection. The TNFR-2W group received delayed TNFR surgery 2 weeks after nerve transection (Day 14), while the TNFR-4W group received delayed TNFR surgery 4 weeks after nerve transection (Day 28). Functional and histological evaluations were conducted at 4 weeks post-TNFR surgery for all groups. 2.2 Surgical procedures A TNFR model was established following the TMR surgery protocol described by Jianping Huang et al. [ 22 ]. Rats were deprived of food for 12 hours and water for 4 hours prior to surgery. This fasting protocol was implemented to minimize the risk of aspiration during anesthesia, a standard precaution in rodent surgical procedures. Perioperative analgesia was administered to ensure animal welfare. Buprenorphine (0.05 mg/kg) was given subcutaneously 30 minutes prior to surgery to manage pain and provide prolonged analgesic effects. Additional doses were administered every 12 hours postoperatively for 48 hours to ensure adequate pain relief during the recovery period. Anesthesia was induced with 3% isoflurane for approximately 3–5 minutes and maintained at 2% isoflurane throughout the operation. The anesthetized rats were fixed in a supine position on the operating table, and the surgical area was fully exposed and depilated for skin preparation. The right ventral side of each group of rats was used as the experimental side. The right median nerve was anastomosed with the musculocutaneous nerve, and recording electrodes were implanted in the biceps brachii. The left ventral side was used as the normal side, only the recording electrodes were implanted, and the rest was left untreated.The area was disinfected with povidone-iodine and then deiodinated with 75% alcohol after drying. A surgical incision was made 1.5-2 cm perpendicular to the midline of the elbow from the acromion. The skin, superficial fascia (subcutaneous tissue), and deep fascia were sequentially opened to isolate the musculocutaneous nerve and the connective tissue adjacent to the median nerve. Once the nerve was fully exposed, the proximal end of the musculocutaneous nerve and the distal end of the median nerve were ligated with an 8 − 0 suture and severed. An end-to-end anastomosis was performed as close as possible to the point where the biceps nerve enters the muscleFollowing nerve anastomosis, we disinfected and sutured the surgical area in layers (Fig. 2 ). Meanwhile, in the control group, only the nerve was exposed, and in the denervated group, only denervation (without anastomosis) was conducted, while in the TNFR-2W group, anastomosis was conducted 2 weeks post-denervation, and in the TNFR-4W group, it was conducted 4 weeks post-denervation. 2.2.1 Implantation of EMG Electrodes in Rats We surgically implanted EMG electrodes into the rats' bilateral biceps brachii muscles. Each rat was positioned prone and secured using a stereotactic device, with an incision made to expose the skull and remove the fascia. A stereotactic device was then implanted and anchored in the skull of each rat to ensure precise and stable placement of electrodes for chronic neural and muscle activity recordings. This setup allows for accurate targeting of specific anatomical sites and minimizes movement-related artifacts, which is essential for obtaining reliable, consistent data across experimental sessions. By securing the device within the skull, we prevented any shifting or dislodgement, which could compromise data quality and animal safety. A precise 1 mm hole was drilled near the 'person' seam, into which a skull nail was inserted, followed by a five-channel connector pre-welded to Teflon-coated stainless steel electrode wires. This assembly was affixed to the skull using ultraviolet-curing adhesive (Fig. 3 ). Subsequently, a transverse incision above the biceps brachii muscles facilitated access, allowing for careful dissection and exposure of the muscle bellies down to the elbow joint. Surgical forceps were used to subcutaneously route the electrode wires from the skull joint to each biceps brachii muscle, with approximately 5 mm of Teflon coating stripped from the wire ends before meticulous insertion into the muscle bellies. The electrodes were secured using a No. 8 suture needle, and the procedure was concluded with suturing of the incisions and administration of penicillin for anti-inflammatory purposes (Fig. 4 ). 2.3 EMG Signal Acquisition and Recording EMG signals play a crucial role in evaluating surgical outcomes and assessing functional recovery. Following established methodologies for intramuscular EMG recording and analysis [ 23 , 24 ], intramuscular EMG signals were recorded using bipolar configuration. Our approach incorporated techniques similar to those described by Boeltz et al. [ 25 ] for recording signals from reinnervated muscles, with signal processing methods adapted from studies on EMG analysis in neuromuscular assessment [ 26 – 28 ]. A five-channel connector (Omnetic, Minneapolis, MN, USA) was fixed on the skull with skull nails. Teflon-coated stainless steel wires (Cat# 793500, A-M System Inc, Sequim, WA, USA) were passed subcutaneously to the back (grounded electrode) and both biceps brachii (recording electrodes). After exposing the muscles bilaterally through transverse skin incisions, a 3 mm notch was made in the Teflon coating of the electrodes before implantation. Intramuscular myoelectric signals were collected for 4 weeks starting 1 week after the experimental operation. The electrical stimulation was performed using an invasive approach, with direct surgical exposure of the target median nerve. The EMG signal data were acquired using a self-developed multichannel biopotential signal system at a sampling rate of 1024 Hz (NES-128B01, 128 channels, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China). During the EMG recording, rats performed locomotion on a self-developed wheel treadmill (diameter, 0.4 m; circumference, 1.26 m; maximum speed of 30 r/min [0.63 m/s]). For formal experiments, the parameters were set as follows: running time, 30 seconds; rest time, 30 seconds; number of cycles, 3 (total time, 3 minutes); and speed, 9 r/min (0.19 m/s) (Fig. 5 ). 2.4 EMG Signal Processing and Analysis The analysis methods were designed to comprehensively assess the intensity, power, and frequency characteristics of EMG signals. First, raw EMG signals were preprocessed by applying a 4th-order Butterworth bandpass filter (20–150 Hz) directly in the time domain to remove motion artifacts and high-frequency noise while preserving physiologically relevant components. This approach was selected over frequency domain filtering as it is more appropriate for real-time bioelectrical signal processing and has become standard practice in EMG analysis. A dual approach combining Short-Time Fourier Transform (STFT) for time-frequency analysis and Root Mean Square (RMS) value calculations was implemented. This approach captures frequency variations over time, providing insights into the dynamic nature of muscle activity post-surgery. The amplitude was derived as the modulus (absolute value) of the STFT results: The parameters for the Short-Time Fourier Transform (STFT) were chosen thoughtfully to enhance the analysis of the signal characteristics. The window function employed was the Hamming window, chosen for its optimal trade-off between main-lobe width (3.3 dB) and side-lobe suppression (-42 dB), minimizing spectral leakage compared to rectangular windows. Window length was set to 256 samples (250 ms at 1024 Hz sampling rate). This duration captures 5 cycles of the lowest frequency component (20 Hz) while maintaining quasi-stationarity within each window. Overlap of 50% (128 samples) was implemented to reduce edge effects and improve temporal resolution to 125 ms. Additionally, zero-padding to 512 points enhanced the frequency resolution to 2 Hz. The STFT was computed using MATLAB's spectrogram function (Signal Processing Toolbox, R2020a) with the aforementioned parameters. The RMS value was utilized to quantify the total energy of the EMG)signal, integrating both amplitude and contraction duration: $$\:\text{RMS}\text{}=\sqrt{\frac{1}{N}\sum\:_{i=1}^{N}\left({x}_{i}^{2}\right)}$$ 3 where N represents the total number of samples in each 1-second window (1024 samples at our sampling rate), and \(\:x\_i\) represents the amplitude value of the EMG signal at sample i (unit: µV). For all analyses, we specifically selected the central 20-second period of EMG activity from each 30-second running period to avoid transitional effects at the beginning and end of each running cycle. Within this 20-second stable activity period, RMS values were calculated from consecutive 1-second segments with 50% overlap (0.5-second step size), resulting in 39 overlapping windows for each 20-second period. For each rat, we computed the mean of these RMS values across all analyzed segments within each recording session, providing a single representative measure of muscle activity intensity for statistical comparison between experimental groups. Additionally, we calculated the standard deviation of RMS values to assess the variability of muscle activity within each session. The frequency band of 40–80 Hz was specifically monitored in the STFT results, as this mid-frequency range has been shown to be most sensitive to changes in motor unit recruitment patterns during recovery [ 29 – 31 ]. All signal processing and analyses were performed using MATLAB software (Matlab R2020, MathWorks, Natick, MA, USA). In Fig. 5 B and 5 C, the x-axis represents time (milliseconds) and the y-axis represents amplitude (microvolts). The combination of amplitude and RMS provides a balanced assessment of muscle activity, capturing both the magnitude and power of the EMG signals. This dual approach ensures a thorough evaluation of muscle function under different experimental conditions. 2.5 Measurement of behavioral function The post-surgery recovery of phantom limb pain in rats was observed through their autophagy behavior. After collecting intramuscular EMG data, we observed the rats daily for self-injurious behavior. The degree of autophagy in toes is evaluated using a scoring system where a maximum of 13 points can be assigned [ 32 ] (Table 1 ). Table 1 Autophagy Scoring System One point is awarded for damage to one or more toenails; one point is given for damage to the distal segment of each toe, with each affected toe contributing one point; if autophagy occurs in the distal segment of all toes, one point is awarded in total regardless of the number of toes affected; one point is given for damage to the proximal phalangeal segment of each toe, with each affected toe contributing one point; and if autophagy occurs in the proximal phalangeal segment of all toes, one point is awarded in total regardless of the number of toes affected. This system ensures consistent point allocation based on the specific damage or autophagy observed. 2.6 In vivo mechanical analysis of biceps brachii The in vivo mechanical analysis of the biceps brachii muscle provides valuable insights into its functionality under both normal and pathological conditions, and helps in assessing the efficacy of various treatments, including post-surgical recovery [ 33 ]. Following the observation of autophagic behaviors, we conducted an examination of the biomechanical properties of the biceps brachii muscle using the Melab-u/8c502 biosignal acquisition system, equipped with a pressure sensor capable of handling up to 50 grams (Fig. 6 ). To ensure accurate measurements, we stimulated the proximal region of the nerve graft. This allowed us to evaluate the functional mimicry between the median nerve and the musculocutaneous nerve subsequent to surgical interventions. 2.7 Hematoxylin and eosin (H.E.) staining of the DRG After measuring muscle strength, morphological observations were conducted using Hematoxylin and Eosin (H.E.) staining to examine the dorsal root ganglia (DRG), which are critical structures where sensory neurons converge. This technique allowed us to identify and assess the characteristics of these neurons within the ganglia. Following the in vivo mechanical analysis, the rats were anesthetized with isoflurane and cannulated through the left ventricle to the ascending aorta for perfusion fixation. The spinal cord and DRG of the C5-T1 segment were removed and immediately fixed in 4% paraformaldehyde for 24 hours. The samples were dehydrated using a gradient sucrose solution, embedded in an optimal cutting temperature compound, and stored at -80 ℃. The embedded DRG samples were then sectioned at 20µm thickness using a Leica CM1950 cryostat, and stained with H.E. stain (ZSGBBio, Beijing, China). The slides were observed under an upright microscope (Eclipse E100, Nikon, Japan) with a 40× objective lens, and the number of neurons was counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.8 Immunofluorescence staining of the spinal cord Finally, the spinal cord was subjected to immunofluorescence staining to assess the distribution and morphology of synapses on motor neurons. Synaptophysin (SYN) and postsynaptic density protein 95 (PSD-95) are pivotal proteins that modulate neuromuscular junction function and play crucial roles in nerve signal transmission and muscle action [ 34 ]. Spinal cord tissues embedded in paraffin were sectioned at 15–20 µm thickness using a Leica cryostat at -20 ℃. These sections were then deparaffinized, rehydrated, and subjected to antigen retrieval. The sections were blocked with bovine serum albumin and incubated with primary antibodies against SYN (rabbit anti-SYN antibody, 1:500, Cat# bs-23504R, RRID: AB_2895150, Bioss) and PSD-95 (mouse-produced primary antibody, specific catalog and concentration to be added). Following primary antibody incubation, the sections were incubated with fluorescently labeled secondary antibodies: goat anti-rabbit IgG/Cy3 for SYN (green fluorescence) and goat anti-mouse IgG conjugated to Alexa Fluor 594 for PSD-95 (red fluorescence). After nuclear counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI, Abcam), the sections were mounted and observed under an upright fluorescence microscope (E100, Nikon) using a 40x objective lens. The mean optical density of SYN and PSD-95 was quantified using ImageJ software (National Institutes of Health). 2.9 Statistical analysis Functional and histopathological analyses were performed under blinding protocols to ensure unbiased results. The normality test was performed using the Shapiro-Wilk test with a significance level of 0.05. Homogeneity of variances was confirmed with Levene's test. All data samples conformed to normal distributions (P < 0.05). We present quantitative data as means ± standard deviation. Differences between experimental groups were assessed using one-way ANOVA, followed by post-hoc Bonferroni tests for all multiple comparisons. This approach was selected to control for Type I error rates across all analyses. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were conducted under blinding protocols using GraphPad Prism software version 9.0.0 (GraphPad Software, San Diego, CA, USA). 3. Results 3.1 General conditions of rats Post-surgery, all rats exhibited normal feeding behavior and showed no signs of infections, ulcers, or nervous system disorders. 3.2 Effect of TNFR surgery on intramuscular EMG signal analysis in rats EMG signals were successfully collected from rats 4 weeks post-surgery. Figures 7A-E and 8A-E show the typical averaged waveforms and muscle activity spectra of EMG signals from all groups (control, denervation, TNFR, TNFR-2W, and TNFR-4W), respectively. The biceps brachii muscles of the TNFR, TNFR-2W, and TNFR-4W groups produced EMG signals of varying amplitudes post-TNFR surgery (Figs. 7B-D and 8B-D). The amplitude and muscle activity of EMG signals in the TNFR group were significantly higher than those observed in all other experimental groups. In stark contrast, the denervated group exhibited only background noise with no detectable EMG signals (Figs. 7E and 8E), confirming complete loss of neuromuscular activity. The comprehensive statistical analysis of intramuscular signal parameters across all experimental conditions is presented in Figs. 7F and 8F, which display the quantitative amplitude and RMS values, respectively. These analyses clearly demonstrate the superior functional recovery achieved with immediate TNFR intervention compared to delayed procedures. Significant differences were observed in the amplitude and RMS values among the groups (Figs. 7F and 8F). The amplitude and RMS values were significantly higher in the control group compared to the TNFR group (P < 0.05). Within the TNFR groups, the amplitude and RMS values were higher in the TNFR group than in the TNFR-2W and TNFR-4W groups (P < 0.05). Amplitude and RMS values were significantly lower in the denervation group compared to the other groups (P 0.05), both groups exhibited amplitude and RMS values significantly higher than those of the denervation group (P < 0.05). 3.3 Autotomy behavior observation The experimental results indicated that the control and TNFR groups displayed no abnormalities in toe morphology. However, in the denervation group, phantom limb pain and abnormal autophagy behavior were observed as early as one week post-surgery, with symptoms intensifying over time (Fig. 9). The TNFR-2W and TNFR-4W groups exhibited autophagy, redness, and swelling of the toes at one week post-surgery, but these symptoms, along with the phantom limb pain, decreased and eventually subsided within 14–21 days. Table 2 shows the autotomy behavior in rats across different time points for each experimental group. In the TNFR-2W group, 3 rats exhibited level 1 autotomy behavior in Week 1 while 3 had no symptoms; 4 rats showed level 2 behavior in Week 2 with 2 remaining asymptomatic; by Week 3, only 2 rats still displayed level 1 behavior with 4 recovered to no symptoms; and by Week 4, all 6 rats had completely recovered with no symptoms. The TNFR-4W group showed a similar recovery pattern, with 3 rats displaying level 1 behavior in Week 1, progressing to 4 rats with level 2 behavior in Week 2, then improving to 4 rats with level 1 behavior in Week 3, and finally all 6 rats showing complete recovery by Week 4. In contrast, rats in the denervation group demonstrated progressively worsening symptoms, with 5 rats showing level 1 autotomy in Week 1, all 6 rats reaching level 2 in Week 2, and all 6 rats progressing to severe level 3 autotomy behavior by Weeks 3 and 4. This demonstrates that without intervention, denervated rats experienced continual deterioration in symptoms throughout the observation period. Additionally, by the end of the observation period, the toe morphologyin the TNFR intervention groups (TNFR-2W and TNFR-4W) appeared similar to that observed in the control group, suggesting resolution of the initial autotomy symptoms. This demonstrates that without intervention, denervated rats experienced continual deterioration in symptoms throughout the observation period. Table 2 Autophagy of rats in each group. The numbers in parentheses represent the number of animals exhibiting autophagy behavior in each group per week. Groups Symptom Level Week1 Week 2 Week 3 Week 4 Control group No symptoms 6 6 6 6 Level 1 (+) 0 0 0 0 Level 2 (++) 0 0 0 0 Level 3 (+++) 0 0 0 0 TNFR group No symptoms 6 6 6 6 Level 1 (+) 0 0 0 0 Level 2 (++) 0 0 0 0 Level 3 (+++) 0 0 0 0 TNFR-2W No symptoms 3 2 4 6 Level 1 (+) 3 0 2 0 Level 2 (++) 0 4 0 0 Level 3 (+++) 0 0 0 0 TNFR-4W No symptoms 3 2 2 6 Level 1 (+) 3 0 4 0 Level 2 (++) 0 4 0 0 Level 3 (+++) 0 0 0 0 Denervated No symptoms 1 0 0 0 Level 1 (+) 5 0 0 0 Level 2 (++) 0 6 0 0 Level 3 (+++) 0 0 6 6 TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, and TNFR-4W: 4-week delayed TNFR group 3.4 Effect of TNFR surgery on biceps brachii contraction force Four weeks post-surgery, both the maximum contraction force and the maximum tetanic contraction force of the biceps brachii were significantly higher in the control group compared to the denervation, TNFR, TNFR-2W, and TNFR-4W groups (P < 0.05) (Fig. 10). Furthermore, the TNFR group showed significantly greater muscle strength than the TNFR-2W, TNFR-4W, and denervation groups (P 0.05), though both were significantly stronger than the denervation group (P < 0.05). 3.5 Effects of TNFR surgery on the DRG neurons in rats H.E. staining was performed on the DRG samples of each group. The number of sensory neurons was significantly higher in the TNFR group than in the TNFR-2W and TNFR-4W groups (P < 0.05; Fig. 11). The TNFR-2W group showed a trend toward better preservation of neuronal morphology compared to the TNFR-4W and denervation groups, although this difference was not statistically significant (P > 0.05) 3.6 Effects of TNFR surgery on SYN and PSD-95 expression in the spinal cord motor neurons in rats Immunoreactivity in the motor neurons of the anterior horn of the spinal cord in control group rats. Compared with the control group, the SYN immunoreactivity in the TNFR group showed a reduction (P > 0.05; Fig. 12P). The SYN immunoreactivity was significantly higher in the TNFR group compared to the TNFR-2W, TNFR-4W, and denervation groups (P < 0.05; Fig. 12P), demonstrating better preservation of presynaptic terminals with immediate intervention. For PSD-95 expression, all experimental groups (TNFR, TNFR-2W, TNFR-4W, and denervation groups) showed significantly reduced levels compared to the control group (P 0.05; Fig. 12Q), indicating that intervention timing had no differential impact on postsynaptic protein expression. While fluorescence intensity provides valuable quantitative and spatial information on protein expression, it may be influenced by external factors such as staining time and antibody concentration. To mitigate these potential variables, we implemented rigorous methodological controls including standardized staining protocols, consistent antibody lots, uniform incubation times, and calibrated image acquisition settings across all experimental groups. This standardization ensures that the observed differences in SYN and PSD-95 expression patterns (Fig. 12) accurately reflect biological changes rather than technical variations. Representative immunofluorescence staining images of SYN/PSD-95 in the control (A), denervated (B), TNFR (C), TNFR-2W (D), and TNFR-4W (E) groups ( scale bar: 200 µm, original magnification 4×). (F-J) Magnified images of the SYN/PSD-95-stained areas in A-E (scale bar: 50 µm, original magnification 10×). (K-O) Merged images showing co-localization. Quantitative analysis of (P) relative fluorescence intensity of SYN and (Q) relative fluorescence intensity of PSD-95 across groups. Data are expressed as mean ± SD, n = 6 per group. Statistical analysis: one-way ANOVA (SYN: F (4, 25) = 18.72, P < 0.0001; PSD-95: F (4, 25) = 31.24, P < 0.0001) followed by Bonferroni multiple comparisons test. *indicates the difference between the control group and other groups, *P < 0.05; #indicates the difference between TNFR and other groups, #P < 0.05. SYN: synaptophysin, PSD-95: post-synaptic density protein 95, TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation 4. Discussion In this experiment, we demonstrated that immediate nerve reinnervation following injury significantly improves muscle function, showing markedly better results compared to reinnervation delayed by 2 and 4 weeks. Additionally, we found that combining implanted electrodes with TNFR is an effective method for in vivo tracking of functional recovery. Our research confirms the feasibility of functional restoration through TNFR surgery, supported by both bio-structural and EMG data as shown in Figs. 7 and 8 , which is crucial for the development of myoelectric prosthetics. Early detection of signals from the target muscle post-surgery indicates a significant reduction in overall functional recovery time. These findings provide important evidence for further optimizing nerve reinnervation surgery. TMR can significantly enhance axon regeneration, increase the quantity and size of regenerated axons, shorten the duration of muscle reinnervation, and prevent neuromas [ 35 – 37 ]. In contrast, our TNFR approach offers distinct advantages by maintaining original neural pathways while providing supplementary neural input. In this work, we strategically placed the site for neurorrhaphy close to the target muscle to reduce both the regeneration distance and duration as illustrated in Fig. 4 . To achieve superior results, we made several refinements to the experimental protocol. Initially, we used a pull hook to elevate the pectoralis major, pectoralis minor, and portions of the deltoid. Although this could have prevented muscle trauma, it did not provide a satisfactory view of the operative site. Therefore, we opted to carefully separate the pectoralis major and minor muscles to obtain a clear view of the operation site, reducing surgical time despite its unfavorable effect on motor function. Regarding the surgical approach, following guidelines from a previous study [ 38 ], we ensured optimal conditions for nerve reinnervation. By incising the musculocutaneous nerve (MCN) proximally and the median nerve (MN) distally, we increased nerve length, facilitating a tension-free neurorrhaphy as shown in Fig. 2 C. We refined our surgical procedure to prevent damage to the local vascular network and limit nerve disconnection to only the necessary segments for ligation. This approach effectively reduced bleeding and enhanced blood supply to the transected nerve, crucial for delivering nutrients and regeneration factors [ 39 ]. Neovascularization served as a conduit for the restored nerve, directing regenerating axons [ 40 – 42 ] and significantly reducing recovery time. We performed second-stage operations with remarkable precision and ease. The surgical field was unobstructed, allowing for direct and effortless nerve access. This streamlined approach resulted from meticulous planning and execution, ensuring minimal tissue manipulation to effectively expose target areas. Consequently, this may have contributed to reduced fibroblast infiltration and lower damage to blood vessels. For functional assessment, the treadmill examination is a streamlined and precise method to regulate and assess the behavioral parameters of rats during training regimens [ 42 , 43 ]. In this project, we used a customized wheel treadmill as depicted in Fig. 5 A, unlike conventional flat treadmills, to enhance engagement and activity in the forelimbs. Our preliminary tests revealed that the wheel treadmill prevented the rats from leaping forward using their hind limbs, thereby compelling increased reliance on and participation of the forelimbs. After a brief training period, the rats demonstrated improved balance and active participation on the wheel treadmill. This approach provided an effective strategy for observing and assessing the recovery trajectory of forelimb muscle functionality and neural control. To validate this method, we also measured the maximum contraction force and maximum tetanic contraction force of the biceps brachii muscle presented in Fig. 10 A-B, which helped assess the function and health status of the muscle fibers. In the TNFR group, the immediate application of TNFR following an injury resulted in the highest myoelectric signal amplitudes, maximum contraction force, and maximum tetanic contraction force compared with the other experimental groups. In contrast, both signal amplitude and muscle power significantly decreased in the delayed TNFR groups, specifically the TNFR-2W and TNFR-4W groups. Therefore, immediate TNFR preserved significantly better muscle function compared with delayed intervention. Irreversible muscle atrophy post-denervation, as described by Soendenbroe [ 44 ], can lead to severe muscle weakness and unfavorable functional prognoses. This accounts for the poor performance observed in the denervation group during functional assessments, where all three evaluation indices were lower than those of the other groups as demonstrated in Figs. 7 F, 8 F, and 10 A-B. The delayed TNFR groups showed no significant differences in their myoelectric signal patterns and muscle power, consistent with the results of the functional assessment. These results indicate the significant benefits of TNFR, especially immediate TNFR intervention, for effective restoration of motor function. Our findings on the timing-dependent efficacy of nerve reinnervation parallel recent evidence in related fields. A review by Dominguez et al. [ 45 ] demonstrated similar timing-dependent effects with TMR, where acute TMR showed superior pain management outcomes compared to delayed procedures, further supporting the critical importance of early intervention in neural pathway restoration. Regarding behavioral outcomes, while phantom limb pain typically occurs in individuals following limb amputation, the observed self-mutilation behaviors (or autotomy) in our study may indicate neuropathic pain or sensory disturbances associated with delayed nerve repair rather than phantom pain. Autotomy behaviors were observed in rats that underwent delayed surgeries, which involved nerve severance, while those receiving immediate surgery did not exhibit these signs as documented in Fig. 9 and Table 2 . Autotomy behaviors rarely follow median nerve injury [ 46 , 47 ]. We selected Sprague Dawley rats for their availability and established use in nerve injury models. However, Carr et al. [ 48 ], noted this strain shows higher rates of autotomy after injury compared to Lewis rats, which exhibit much lower rates of this behavior. Future studies might consider using alternative strains with lower susceptibility to autotomy to further minimize these effects and clarify if the autophagy behavior observed is strain-specific or model-dependent. These results suggest that immediate TNFR intervention yields the best results in nerve reconstruction. For histological findings, DRG are composed of sensory fiber cells that receive all nerve impulses, including general somatosensory and visceral sensations, from the body's receptors. These impulses are then relayed to the spinal cord via sensory fibers. In this study, we used H.E. staining to examine the morphology and number of sensory neurons in the DRG as shown in Fig. 11 A-O. Our analysis revealed that TNFR intervention effectively normalized neuronal morphology, restoring the size of the cell bodies and the number of axons to levels statistically similar to those in the control group. However, delayed TNFR treatment in the TNFR-2W and TNFR-4W groups only partially recovered sensory neuron morphology and functionality, with observed instances of demyelination. Quantitative analysis (Fig. 11 P) confirmed a significant reduction in sensory neuron numbers in the TNFR-2W and TNFR-4W groups compared to immediate TNFR. Therefore, timely TNFR intervention, particularly when implemented immediately, is essential for the effective recovery of sensory neurons in the DRG. Regarding synaptic marker analysis, SYN protein is localized to synaptic vesicles in the presynaptic terminals of neurons. It is involved in the release of activity-dependent neurotransmitters and plays a crucial role in synaptic plasticity. In contrast, PSD-95 is essential for post-synaptic signal transduction and synaptic plasticity by anchoring receptor proteins at the synaptic membrane [ 49 , 50 ] Alterations in PSD-95 expression in motor neurons can affect synaptic strength, contributing to changes in motor activity. Our results showed a significant decrease in the expression of SYN and PSD-95 across all experimental groups compared to the control group as visualized in Fig. 12 A-O. SYN, a presynaptic vesicle protein essential for neurotransmitter release, showed relatively better restoration in the immediate TNFR group compared to the TNFR-2W, TNFR-4W, and denervated groups. However, PSD-95, a scaffolding protein critical for postsynaptic organization and signaling, remained similarly reduced across all intervention groups as quantified in Fig. 12 P-Q, suggesting differential effects on pre-and postsynaptic markers. This pattern indicates that while immediate TNFR intervention may partially preserve presynaptic structures (as evidenced by improved SYN expression), postsynaptic architecture (represented by PSD-95) appears more resistant to recovery regardless of intervention timing. The persistent reduction in PSD-95 across all experimental groups suggests that postsynaptic densities in motor neurons within the anterior horn of the spinal cord may require additional interventions beyond TNFR to achieve complete recovery. This differential response between presynaptic and postsynaptic markers highlights the complex nature of synaptic reorganization following peripheral nerve injury and suggests that comprehensive neuronal recovery may require targeted approaches that address both pre- and postsynaptic elements of the neural circuit. 5. Limitations First, our rat model may not fully represent the complexity of human peripheral nerve injuries due to anatomical and physiological differences. Second, while we demonstrated functional recovery through EMG and behavioral assessments, the molecular mechanisms underlying TNFR-mediated neuronal survival remain unexplored. Third, the observed autotomy behavior in Sprague Dawley rats may be strain-specific and should be interpreted cautiously when translating to clinical applications. Finally, challenges in maintaining EMG signal quality over extended periods due to potential immune rejection and electrode degradation warrant future development of more biocompatible electrode arrays to enhance signal fidelity for long-term monitoring. 6. Conclusion This study demonstrated that TNFR effectively promotes functional recovery following nerve injury in rats, with immediate intervention yielding significantly better outcomes than delayed procedures. Our comprehensive evaluation revealed superior EMG signals, reduced autophagic behavior, and better preservation of muscle function and neuronal structures with immediate TNFR. These findings highlight the critical importance of early intervention in peripheral nerve injuries and provide valuable insights for improving surgical strategies and neuroprosthetic development in clinical settings. Abbreviations ANOVA: Analysis of Variance ARRIVE: Animal Research: Reporting of In Vivo Experiments DRG: Dorsal Root Ganglion EMG: Electromyography GND: Ground H.E.: Hematoxylin and Eosin MCN: Musculocutaneous Nerve MN: Median Nerve PSD-95: Postsynaptic Density Protein 95 RPNI: Regenerative Peripheral Nerve Interfaces RMS: Root Mean Square SPF: Specific Pathogen-Free STFT: Short-Time Fourier Transform SYN: Synaptophysin TMR: Targeted Muscle Reinnervation TNFR: Targeted Nerve Function Replacement TNFR-2W: 2-Week Delayed Targeted Nerve Function Replacement TNFR-4W: 4-Week Delayed Targeted Nerve Function Replacement Declarations Author contributions The study conception and design were formulated by Chunxiao Tang and Yuanheng Li. Chunxiao Tang conducted the formal analysis, methodology, investigation, and drafted the original manuscript. Yuanheng Li contributed to the investigation, software, and formal analysis. Jiamei Guo and Xinxian Fan assisted with formal analysis, methodology, and investigation. Yifeng Lin was responsible for producing the article illustrations. Yifan Gao and Lin Yang provided supervision, investigation, and editorial support. Funding This work has been supported by grants from National Natural Science Foundation of China (#82260456, 81921804), Science and Technology Planning Project of Shenzhen (JCYJ20230807140559047), Science and Technology Department of Guizhou Province (202342938082710225), Zunyi city science and technology plan project(HZ-2020-56), Zunyi Medical University(F-ZH-015, 2018-5772-063), SIAT College Student Innovation Practice Training Program (2023-38, 2023-20). Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate The study protocol was approved by the Animal Ethics Committee of the Zhuhai campus of Zunyi Medical University, Zhuhai, China (approval No. 2019-2-273) on March 11, 2019. The study was conducted in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) Consent for publication The authors consent to publish. Competing interests The authors declare they do not have any competing interests. References Jensen, S. E., Z. Butt, A. Bill, T. Baker, M. M. Abecassis, A. W. Heinemann, D. Cella, and G. A. Dumanian. "Quality of Life Considerations in Upper Limb Transplantation: Review and Future Directions." J Hand Surg Am 37, no. 10 (2012): 2126-35. Kuiken, T. A., L. A. Miller, R. D. Lipschutz, B. A. Lock, K. Stubblefield, P. D. Marasco, P. Zhou, and G. A. Dumanian. "Targeted Reinnervation for Enhanced Prosthetic Arm Function in a Woman with a Proximal Amputation: A Case Study." Lancet 369, no. 9559 (2007): 371-80. Kuiken, Todd A, Guanglin Li, Blair A Lock, Robert D Lipschutz, Laura A Miller, Kathy A Stubblefield, and Kevin B Englehart. "Targeted Muscle Reinnervation for Real-Time Myoelectric Control of Multifunction Artificial Arms." Jama 301, no. 6 (2009): 619-28. Kuiken, Todd A. "The Scientific Basis of Targeted Muscle Reinnervation." Targeted Muscle Reinnervation: A Neural Interface for Artificial Limbs 9 (2013). Kuiken, T. A., A. K. Barlow, L. Hargrove, and G. A. Dumanian. "Targeted Muscle Reinnervation for the Upper and Lower Extremity." Tech Orthop 32, no. 2 (2017): 109-16. Cheesborough, J. E., L. H. Smith, T. A. Kuiken, and G. A. Dumanian. "Targeted Muscle Reinnervation and Advanced Prosthetic Arms." Semin Plast Surg 29, no. 1 (2015): 62-72. Vadalà, G., G. Di Pino, L. Ambrosio, L. Diaz Balzani, and V. Denaro. "Targeted Muscle Reinnervation for Improved Control of Myoelectric Upper Limb Prostheses." J Biol Regul Homeost Agents 31, no. 4 suppl 1 (2017). Luft, M., J. Klepetko, S. Muceli, J. Ibáñez, V. Tereshenko, C. Festin, G. Laengle, O. Politikou, U. Maierhofer, D. Farina, O. C. Aszmann, and K. D. Bergmeister. "Proof of Concept for Multiple Nerve Transfers to a Single Target Muscle." Elife 10 (2021). Miller, L. A., R. D. Lipschutz, K. A. Stubblefield, B. A. Lock, H. Huang, T. W. Williams, 3rd, R. F. Weir, and T. A. Kuiken. "Control of a Six Degree of Freedom Prosthetic Arm after Targeted Muscle Reinnervation Surgery." Arch Phys Med Rehabil 89, no. 11 (2008): 2057-65. O'Brien, A. L., S. W. Jordan, J. M. West, L. M. Mioton, G. A. Dumanian, and I. L. Valerio. "Targeted Muscle Reinnervation at the Time of Upper-Extremity Amputation for the Treatment of Pain Severity and Symptoms." J Hand Surg Am 46, no. 1 (2021): 72.e1-72.e10. Stoehr, J. R., R. Sood, S. W. Jordan, and G. A. Dumanian. "Targeted Muscle Reinnervation at the Time of Amputation in the Management of Complex Regional Pain Syndrome of the Lower Extremity." Microsurgery 40, no. 8 (2020): 852-58. ElAbd, Rawan, Todd Dow, Sinan Jabori, Becher Alhalabi, Samuel J Lin, and Sammy Dowlatshahi. "Pain and Functional Outcomes Following Targeted Muscle Reinnervation: A Systematic Review." Plastic and Reconstructive Surgery 153, no. 2 (2024): 494-508. Smith, B. W., A. K. Daunter, L. J. Yang, and T. J. Wilson. "An Update on the Management of Neonatal Brachial Plexus Palsy-Replacing Old Paradigms: A Review." JAMA Pediatr 172, no. 6 (2018): 585-91. Leach, G. A., R. A. Dean, N. G. Kumar, C. Tsai, F. E. Chiarappa, P. S. Cederna, T. A. Kung, and C. M. Reid. "Regenerative Peripheral Nerve Interface Surgery: Anatomic and Technical Guide." Plast Reconstr Surg Glob Open 11, no. 7 (2023): e5127. Frost, Christopher M., Daniel C. Ursu, Shane M. Flattery, Andrej Nedic, Cheryl A. Hassett, Jana D. Moon, Patrick J. Buchanan, R. Brent Gillespie, Theodore A. Kung, Stephen W. P. Kemp, Paul S. Cederna, and Melanie G. Urbanchek. "Regenerative Peripheral Nerve Interfaces for Real-Time, Proportional Control of a Neuroprosthetic Hand." Journal of NeuroEngineering and Rehabilitation 15, no. 1 (2018): 108. Wu, Peng. "Delayed Repair of the Peripheral Nerve: A Novel Model in the Rat Sciatic Nerve." Journal of neuroscience methods 214, no. 1 (2013): 37-44. Xu, C., Y. Kou, P. Zhang, N. Han, X. Yin, J. Deng, B. Chen, and B. Jiang. "Electrical Stimulation Promotes Regeneration of Defective Peripheral Nerves after Delayed Repair Intervals Lasting under One Month." PLoS One 9, no. 9 (2014): e105045. Griffin, J. W., M. V. Hogan, A. B. Chhabra, and D. N. Deal. "Peripheral Nerve Repair and Reconstruction." J Bone Joint Surg Am 95, no. 23 (2013): 2144-51. Grinsell, D., and C. P. Keating. "Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies." Biomed Res Int 2014 (2014): 698256. Kubiak, C. A., S. W. P. Kemp, and P. S. Cederna. "Regenerative Peripheral Nerve Interface for Management of Postamputation Neuroma." JAMA Surg 153, no. 7 (2018): 681-82. Percie du Sert, Nathalie, Viki Hurst, Amrita Ahluwalia, Sabina Alam, Marc T. Avey, Monya Baker, William J. Browne, Alejandra Clark, Innes C. Cuthill, Ulrich Dirnagl, Michael Emerson, Paul Garner, Stephen T. Holgate, David W. Howells, Natasha A. Karp, Stanley E. Lazic, Katie Lidster, Catriona J. MacCallum, Malcolm Macleod, Esther J. Pearl, Ole H. Petersen, Frances Rawle, Penny Reynolds, Kieron Rooney, Emily S. Sena, Shai D. Silberberg, Thomas Steckler, and Hanno Würbel. "The Arrive Guidelines 2.0: Updated Guidelines for Reporting Animal Research." BMC Veterinary Research 16, no. 1 (2020): 242. Jianping, Huang, L. I. Wenqing, Yang Lin, Z. H. U. Mingxing, Z. H. U. Xiaodi, L. I. Chuyan, Yang Zijian, and L. I. Guanglin. "A Pilot Study of Nerve Function Reinnervation on a Transhumeral Amputee." Journal of Integration Technology 5, no. 5: 30-37. Citi, L., J. Carpaneto, K. Yoshida, K. P. Hoffmann, K. P. Koch, P. Dario, and S. Micera. "On the Use of Wavelet Denoising and Spike Sorting Techniques to Process Electroneurographic Signals Recorded Using Intraneural Electrodes." J Neurosci Methods 172, no. 2 (2008): 294-302. Zhong, S., Z. Zhang, H. Su, C. Li, Y. Lin, W. Lu, Z. Jiang, and L. Yang. "Efficacy of Biological and Physical Enhancement on Targeted Muscle Reinnervation." Cyborg Bionic Syst 2022 (2022): 9759265. Boeltz, T., M. Ireland, K. Mathis, J. Nicolini, K. Poplavski, S. J. Rose, E. Wilson, and A. W. English. "Effects of Treadmill Training on Functional Recovery Following Peripheral Nerve Injury in Rats." J Neurophysiol 109, no. 11 (2013): 2645-57. Griffin, D., and Lim Jae. "Signal Estimation from Modified Short-Time Fourier Transform." IEEE Transactions on Acoustics, Speech, and Signal Processing 32, no. 2 (1984): 236-43. Costa, M. V., L. A. Pereira, R. S. Oliveira, R. E. Pedro, T. V. Camata, T. Abrao, M. A. Brunetto, and L. R. Altimari. "Fourier and Wavelet Spectral Analysis of Emg Signals in Maximal Constant Load Dynamic Exercise." Annu Int Conf IEEE Eng Med Biol Soc 2010 (2010): 4622-5. da Silva, R. A., C. Larivière, A. B. Arsenault, S. Nadeau, and A. Plamondon. "The Comparison of Wavelet- and Fourier-Based Electromyographic Indices of Back Muscle Fatigue During Dynamic Contractions: Validity and Reliability Results." Electromyogr Clin Neurophysiol 48, no. 3-4 (2008): 147-62. Wakeling, J. M., S. A. Pascual, B. M. Nigg, and V. von Tscharner. "Surface Emg Shows Distinct Populations of Muscle Activity When Measured During Sustained Sub-Maximal Exercise." Eur J Appl Physiol 86, no. 1 (2001): 40-7. Huang, H. J., and D. P. Ferris. "Neural Coupling between Upper and Lower Limbs During Recumbent Stepping." J Appl Physiol (1985) 97, no. 4 (2004): 1299-308. Dideriksen, J. L., D. Farina, and R. M. Enoka. "Influence of Fatigue on the Simulated Relation between the Amplitude of the Surface Electromyogram and Muscle Force." Philos Trans A Math Phys Eng Sci 368, no. 1920 (2010): 2765-81. Whishaw, Ian Q., Valerie Berg dall, and Bryan Kolb. "Chapter 8 - Analysis of Behavior in Laboratory Rats11a Version of This Chapter Previously Appeared In: Whishaw, I.Q. Et Al. (1999). Analysis of Behavior in Laboratory Rodents. In Windhorst, U., Johansson, H. (Eds), Modern Techniques in Neuroscience Research, Pp. 1244–1275, Heidelberg: Springer." In The Laboratory Rat (Second Edition) , edited by Mark A. Suckow, Steven H. Weisbroth and Craig L. Franklin, 191-218. Burlington: Academic Press, 2006. Sporer, M. E., M. Aman, K. D. Bergmeister, D. Depisch, K. M. Scheuba, E. Unger, B. K. Podesser, and O. C. Aszmann. "Experimental Nerve Transfer Model in the Neonatal Rat." Neural Regen Res 17, no. 5 (2022): 1088-95. Lu, Wei, Jian-Ping Li, Zhen-Dong Jiang, Lin Yang, and Xue-Zheng Liu. "Effects of Targeted Muscle Reinnervation on Spinal Cord Motor Neurons in Rats Following Tibial Nerve Transection." Neural Regeneration Research 17, no. 8 (2022). Raasveld, Floris V, Maximilian Mayrhofer-Schmid, Benjamin R Johnston, Barbara Gomez-Eslava, Yannick AJ Hoftiezer, Wen-Chih Liu, Ian L Valerio, and Kyle R Eberlin. "Targeted Muscle Reinnervation at the Time of Amputation to Prevent the Development of Neuropathic Pain." Journal of Plastic, Reconstructive & Aesthetic Surgery (2024). Bishay, Jeremy, Isobel Yeap, and Tim Wang. "The Effectiveness of Targeted Muscle Reinnervation in Reducing Pain and Improving Quality of Life for Patients Following Lower Limb Amputation." Journal of Plastic, Reconstructive & Aesthetic Surgery (2024). Bowen, J Byers, Corinne E Wee, Jaclyn Kalik, and Ian L Valerio. "Targeted Muscle Reinnervation to Improve Pain, Prosthetic Tolerance, and Bioprosthetic Outcomes in the Amputee." Advances in Wound Care 6, no. 8 (2017): 261-67. Kuffler, Damien P., and Christian Foy. "Restoration of Neurological Function Following Peripheral Nerve Trauma." International Journal of Molecular Sciences 21, no. 5 (2020): 1808. Wang, S., X. Liu, and Y. Wang. "Evaluation of Platelet-Rich Plasma Therapy for Peripheral Nerve Regeneration: A Critical Review of Literature." Front Bioeng Biotechnol 10 (2022): 808248. Auger, F. A., L. Gibot, and D. Lacroix. "The Pivotal Role of Vascularization in Tissue Engineering." Annu Rev Biomed Eng 15 (2013): 177-200. Giglia, Giuseppe, Fernando Rosatti, Antonino Giulio Giannone, Giuditta Gambino, Maria Grazia Zizzo, Ada Maria Florena, Pierangelo Sardo, and Francesca Toia. "Vascularized Versus Free Nerve Grafts: An Experimental Study on Rats." Journal of Personalized Medicine 13, no. 12 (2023): 1682. Saffari, T. M., M. Bedar, C. A. Hundepool, A. T. Bishop, and A. Y. Shin. "The Role of Vascularization in Nerve Regeneration of Nerve Graft." Neural Regen Res 15, no. 9 (2020): 1573-79. Xu, Y., Y. Yao, H. Lyu, S. Ng, Y. Xu, W. S. Poon, Y. Zheng, S. Zhang, and X. Hu. "Rehabilitation Effects of Fatigue-Controlled Treadmill Training after Stroke: A Rat Model Study." Front Bioeng Biotechnol 8 (2020): 590013. Soendenbroe, C., M. F. Heisterberg, P. Schjerling, A. Karlsen, M. Kjaer, J. L. Andersen, and A. L. Mackey. "Molecular Indicators of Denervation in Aging Human Skeletal Muscle." Muscle Nerve 60, no. 4 (2019): 453-63. Dominguez, Elizabeth, Allisa Barber, Jose Lucas Zepeda, and Gwendolyn Hoben. "How Timing and Preexisting Pain Affect Outcomes of Tmr: A Systematic Review." Plastic and Aesthetic Research 11, no. 0 (2024): 53. Lauer, H., C. Prahm, J. T. Thiel, J. Kolbenschlag, A. Daigeler, D. Hercher, and J. C. Heinzel. "The Grasping Test Revisited: A Systematic Review of Functional Recovery in Rat Models of Median Nerve Injury." Biomedicines 10, no. 8 (2022). Vela, F. J., G. Martínez-Chacón, A. Ballestín, J. L. Campos, F. M. Sánchez-Margallo, and E. Abellán. "Animal Models Used to Study Direct Peripheral Nerve Repair: A Systematic Review." Neural Regen Res 15, no. 3 (2020): 491-502. Carr, M. M., T. J. Best, S. E. Mackinnon, and P. J. Evans. "Strain Differences in Autotomy in Rats Undergoing Sciatic Nerve Transection or Repair." Ann Plast Surg 28, no. 6 (1992): 538-44. Ferreira, A., and M. Rapoport. "The Synapsins: Beyond the Regulation of Neurotransmitter Release." Cell Mol Life Sci 59, no. 4 (2002): 589-95. Thiel, G. "Synapsin I, Synapsin Ii, and Synaptophysin: Marker Proteins of Synaptic Vesicles." Brain Pathol 3, no. 1 (1993): 87-95. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif RatEMGsignalacquisition.mp4 Cite Share Download PDF Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Journal of NeuroEngineering and Rehabilitation → Version 1 posted Editorial decision: Revision requested 17 May, 2025 Reviews received at journal 30 Apr, 2025 Reviews received at journal 19 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 31 Mar, 2025 Submission checks completed at journal 31 Mar, 2025 First submitted to journal 30 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5460332","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436575299,"identity":"8402a8f2-a7b8-4f0e-8ae7-1ba18ecff2ff","order_by":0,"name":"Chunxiao Tang","email":"","orcid":"","institution":"Zunyi Medical University Zhuhai Campus","correspondingAuthor":false,"prefix":"","firstName":"Chunxiao","middleName":"","lastName":"Tang","suffix":""},{"id":436575300,"identity":"18de2a7c-bf9d-4fe7-8b20-9c33b54c9d85","order_by":1,"name":"Yuanheng Li","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuanheng","middleName":"","lastName":"Li","suffix":""},{"id":436575301,"identity":"e394fb56-45ca-4d37-8f47-1a5c50c1fe21","order_by":2,"name":"Xinxian Fan","email":"","orcid":"","institution":"Shenzhen Technology University","correspondingAuthor":false,"prefix":"","firstName":"Xinxian","middleName":"","lastName":"Fan","suffix":""},{"id":436575302,"identity":"0157b44b-c316-49dd-a714-4f1aba1a2cf3","order_by":3,"name":"Jiamei Guo","email":"","orcid":"","institution":"Shenzhen Technology University","correspondingAuthor":false,"prefix":"","firstName":"Jiamei","middleName":"","lastName":"Guo","suffix":""},{"id":436575303,"identity":"2515062e-8ec3-415b-9a17-364751e4429d","order_by":4,"name":"Yifeng Lin","email":"","orcid":"","institution":"Zunyi Medical University Zhuhai Campus","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Lin","suffix":""},{"id":436575304,"identity":"ef6326b6-44b6-44fa-a52c-8c5d012e2442","order_by":5,"name":"Yifan Gao","email":"","orcid":"","institution":"Zunyi Medical University Zhuhai Campus","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Gao","suffix":""},{"id":436575305,"identity":"83672527-6858-49c2-a89d-7ac39e7b8d6e","order_by":6,"name":"Lin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACZhBRYCHHwM7YQIoWAwljBmaitTBAtCQ2MBOrmL+dx/gzj4FEen8zc5s07w4GeX6xA/i1SBzmMTAGasmdcZgRqOUMg+HM2QkErAFqSc4BamkAa2ljSDC4TUCLPFDLYaCWdHmitRgc5jFsBmpJMCBai+FhtmLmPwYShhsPMzZbzm2TIOwXufOHN3+cUWEjL3e8/eGNt2028vzSBLQwMHAYwFgsEsAQJKQcBNgfwFjMH4hRPwpGwSgYBSMPAABp1Dke0x4LLwAAAABJRU5ErkJggg==","orcid":"","institution":"Zunyi Medical University Zhuhai Campus","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-11-15 12:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5460332/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5460332/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12984-025-01666-0","type":"published","date":"2025-07-04T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79802845,"identity":"fd106807-952b-4e99-acf4-83dbd5e5cad1","added_by":"auto","created_at":"2025-04-03 04:31:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195194,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental timeline comparing the effects of TNFR therapy timing on nerve regeneration.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/e566c9159ed280e55042661a.png"},{"id":79802821,"identity":"3e300fae-4246-476f-b125-b953bf7a866d","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188556,"visible":true,"origin":"","legend":"\u003cp\u003eSurgical procedures for the TNFR model in rats\u003c/p\u003e\n\u003cp\u003e(A) Anatomical diagram showing the location of the median nerve and musculocutaneous nerve in the ventral right forelimb of the rat. (B) Nerve transection surgery showing complete transection of the proximal musculocutaneous nerve and the distal median nerve. (C) Nerve suture surgery showing the end-to-end anastomosis between the proximal musculocutaneous nerve and the distal median nerve to establish the TNFR model.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/22f67e3c675c9c7ea668f0e7.png"},{"id":79802811,"identity":"c53c996d-20b3-431c-a1e4-37428ee0bfaf","added_by":"auto","created_at":"2025-04-03 04:31:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":142214,"visible":true,"origin":"","legend":"\u003cp\u003eExternal skull joint fixation\u003c/p\u003e\n\u003cp\u003e(A1 and A2) Separation and suture of the nerves; (B1) Biceps exposure and electrode penetration; (B2) Recording electrode implantation; (C1) Skull exposure and skull nail fixation; (C2) Skull joint fixation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/d54e688a9471867610370d29.png"},{"id":79802818,"identity":"3e774877-b365-438f-9f87-c8a06f3b2a95","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267939,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the experimental design and EMG recording setup\u003c/p\u003e\n\u003cp\u003e(A) Denervation procedure illustrating the dissociated proximal end of the musculocutaneous nerve and distal end of the median nerve on the surgical side, with intact neural anatomy on the normal side. (B) TNFR model showing the end-to-end anastomosis between the proximal musculocutaneous nerve and distal median nerve on the surgical side, with bilateral electrode placement in the biceps brachii muscles. (C) EMG recording configuration depicting the 5-channel connector secured to the skull, bilateral muscle electrodes, ground (GND) electrode placement, and connection to the electromyographic collector, shown from the dorsal perspective.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/85be57bddb98e9e965cf01a6.png"},{"id":79802815,"identity":"3f7854a8-9c7e-4459-a891-d205c026cfb5","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341269,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of an animal treadmill\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of an animal treadmill. The diagram illustrates a rat walking on the treadmill while connected to a self-developed five-channel EMG signal acquisition module via electrode wires implanted in the biceps brachii muscle. The treadmill setup is designed to measure EMG signals during exercise to evaluate muscle function recovery following TNFR surgery. (B) The raw EMG signal recorded from the biceps brachii muscle during treadmill exercise. (C) The filtered EMG signal, showing noise reduction and improved signal clarity after processing. To enhance visualization of waveform details that appear compressed in the full-scale trace, the 500-1000 ms time epoch was extracted and displayed in the inset.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/0ac13d5d01ddf70ce4536bbc.png"},{"id":79803209,"identity":"eed9ef8d-64f3-4144-a228-35d103b37d4b","added_by":"auto","created_at":"2025-04-03 04:39:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":144071,"visible":true,"origin":"","legend":"\u003cp\u003eMuscle strength acquisition system\u003c/p\u003e\n\u003cp\u003eThe biceps brachii on the surgical side was stretched and stimulated via the median nerve, with muscle strength recorded under parameters for maximum contraction force (automatic amplitude adjustment, 1-second main cycle, 0.2 ms pulse width, 0 to 10V amplitude range) and maximum tetanic contraction (automatic frequency adjustment, 1-second main cycle, 0.2 ms pulse width, 5V initial amplitude, 1 to 50 Hz frequency range).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/1ce3c5cc04558cb8e95c04cc.png"},{"id":79802817,"identity":"ae5596df-78d9-491b-bc8b-13ab6fe519b1","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":827022,"visible":true,"origin":"","legend":"\u003cp\u003eIntramuscular EMG signal amplitude analysis across experimental groups\u003c/p\u003e\n\u003cp\u003eRepresentative EMG signal recordings for each experimental group 4 weeks after intervention are shown in 2D time-amplitude plots (top) and 3D spectrograms (bottom): (A) The control group with normal innervation showed robust, high-amplitude EMG activity; (B) the immediate TNFR group showed a significant recovery of the EMG signal pattern; (C) the TNFR-2W group showed moderate recovery with a 2-week delay in intervention; (D) the TNFR-4W group showed similar moderate recovery despite a 4-week delay in intervention; (E) the denervated group showed only background electrical noise with no functional EMG activity. (F) Quantitative comparison of EMG signal amplitudes among groups. Data are presented as mean ± SD (n = 6 per group). Statistical analysis: One-way ANOVA (F (4, 70) = 16.40, P \u0026lt; 0.0001) followed by Bonferroni multiple comparison test. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compared with the control group; #P \u0026lt; 0.01, ##P \u0026lt; 0.0001 compared with the TNFR group. TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/f4a2813a130115913dbc56c7.png"},{"id":79802826,"identity":"9f6d70aa-c813-4719-b2ef-47c753f3f1bf","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":722667,"visible":true,"origin":"","legend":"\u003cp\u003eEMG signal power analysis across experimental groups\u003c/p\u003e\n\u003cp\u003e(A-E) Representative EMG power spectra from each experimental group at 4 weeks post-intervention, displayed as both 3D frequency-power representations (top) and 2D frequency-power plots (bottom): (A) Control group showing robust muscle activity with high power across frequency spectrum; (B) TNFR group demonstrating reduced but substantial muscle activity with moderately preserved power distribution; (C) and (D) TNFR-2W and TNFR-4W groups showing significantly diminished muscle activity with limited power across frequencies; (E) Denervation group displaying only baseline noise without discernible muscle activity patterns. (F) Quantitative comparison of EMG signal RMS values across groups. Data are expressed as mean \u0026nbsp;± SD (n = 6 per group). Statistical analysis: one-way ANOVA (F (4, 70) = 8.886, P \u0026lt; 0.0001) followed by Bonferroni multiple comparisons test. *P \u0026lt; 0.0001 compared to control group; #P \u0026lt; 0.0001 compared to TNFR group. TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/5c56507aaeae8ea625d97e91.png"},{"id":79803515,"identity":"e9e3fef0-e82f-45eb-9cd6-4a8897a75fff","added_by":"auto","created_at":"2025-04-03 04:47:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":97370,"visible":true,"origin":"","legend":"\u003cp\u003eConditions of the autophagic upper limbs in rats\u003c/p\u003e\n\u003cp\u003e(A) Control group showed no autophagy; (B) TNFR group showed no autophagy; (C) denervated group showed level 4 (extremely severe) autophagy; (D) TNFR-2W group showed level 1 (mild) autophagy; and (E) TNFR-4W groups showed level 1 (mild) autophagy. TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, and TNFR-4W: 4-week delayed TNFR group\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/5ece6fc1851a98c4b78c723a.png"},{"id":79802839,"identity":"f08f25de-6826-490c-87cc-2584d75fbc54","added_by":"auto","created_at":"2025-04-03 04:31:31","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":59518,"visible":true,"origin":"","legend":"\u003cp\u003eBiceps contraction force of each group\u003c/p\u003e\n\u003cp\u003eMaximum single contraction force (A) and maximum tetanic contraction force (B) across all experimental groups. Data are expressed as mean ± SD, n = 6 per group. Statistical analysis: one-way ANOVA (F (4, 25) = 31.24, P \u0026lt; 0.01) followed by Bonferroni multiple comparisons test. *indicates the difference between the control group and other groups, *P \u0026lt; 0.05; #indicates the difference between TNFR and other groups, #P \u0026lt; 0.01. TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/91ce7079f5038184376babe2.png"},{"id":79803217,"identity":"4bd2eab0-82e3-4ddb-825c-f9ffab94dbdc","added_by":"auto","created_at":"2025-04-03 04:39:31","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":223127,"visible":true,"origin":"","legend":"\u003cp\u003eE. staining of DRG of C5-T1 segment of the left forelimb in all the groups\u003c/p\u003e\n\u003cp\u003e(A-E) DRG sections at 4× magnification (scale bar: 500 μm): (A) Control, (B) Denervated, (C) TNFR, (D) TNFR-2W, and (E) TNFR-4W groups. (F-J) Magnified images (10×) of the local areas in A-E (scale bar: 200 μm). (K-O) Magnified images (40×) of the local areas in F-J (scale bar: 50 μm). (P) Quantitative analysis of the number of sensory neurons across groups. Data are expressed as mean ± SD, n = 6 per group. Statistical analysis: one-way ANOVA (F (4, 25) =13.64, P \u0026lt; 0.05) followed by Bonferroni multiple comparisons test. *indicates the difference between the control group and other groups, *P \u0026lt; 0.05; #indicates the difference between TNFR and other groups, #P \u0026lt; 0.05. TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/8b8d32748a50c017a4935a63.png"},{"id":79804139,"identity":"e5a2c0a6-3da5-491a-bbca-d6886b265a1c","added_by":"auto","created_at":"2025-04-03 04:55:32","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":597812,"visible":true,"origin":"","legend":"\u003cp\u003eSYN and PSD-95 expression in the motor neurons in the anterior horn of the spinal cord in rats\u003c/p\u003e\n\u003cp\u003eRepresentative immunofluorescence staining images of SYN/PSD-95 in the control (A), denervated (B), TNFR (C), TNFR-2W (D), and TNFR-4W (E) groups ( scale bar: 200 μm, original magnification 4×). (F-J) Magnified images of the SYN/PSD-95-stained areas in A-E (scale bar: 50 μm, original magnification 10×). (K-O) Merged images showing co-localization. Quantitative analysis of (P) relative fluorescence intensity of SYN and (Q) relative fluorescence intensity of PSD-95 across groups. Data are expressed as mean ± SD, n = 6 per group. Statistical analysis: one-way ANOVA (SYN: F (4, 25) = 18.72, P \u0026lt; 0.0001; PSD-95: F (4, 25) = 31.24, P \u0026lt; 0.0001) followed by Bonferroni multiple comparisons test. *indicates the difference between the control group and other groups, *P \u0026lt; 0.05; #indicates the difference between TNFR and other groups, #P \u0026lt; 0.05. SYN: synaptophysin, PSD-95: post-synaptic density protein 95, TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/59657994b06e1ae4765eddad.png"},{"id":86179023,"identity":"4c4e3466-23d8-4d04-a672-d5e337851a24","added_by":"auto","created_at":"2025-07-07 16:14:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4911692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/3dce8666-61a1-47c0-859b-b555b92489d8.pdf"},{"id":79803211,"identity":"afc39e9c-ff7a-45f3-ac7f-8e2a915204f3","added_by":"auto","created_at":"2025-04-03 04:39:31","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2618143,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/721785fe1d3dd6335617caf1.tif"},{"id":79802876,"identity":"9f32250d-9f46-4e69-acff-bd1fc4fb2858","added_by":"auto","created_at":"2025-04-03 04:31:32","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12766336,"visible":true,"origin":"","legend":"","description":"","filename":"RatEMGsignalacquisition.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5460332/v1/44c4427748c2f58a7b4ddabd.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improving Nerve and Muscle Function: An Exploration of Targeted Nerve Function Replacement Following Differential Delay Periods in a Rat Model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMaintaining limb functionality is essential for seamless interaction with the environment. However, traumatic injuries, vascular diseases, and neurological disorders can severely impair mobility, leading to chronic pain and psychological distress [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Current commercial prosthetics often suffer from limited functionality and delayed response due to inadequate electromyography (EMG) signal sources, resulting in awkward and inefficient movements. This highlights the urgent need for multifunctional prosthetics with intuitive control, particularly for high-level amputees, which can be achieved through advanced surgical interventions.\u003c/p\u003e \u003cp\u003eKuiken et al. introduced a novel surgical technique known as Targeted Muscle Reinnervation (TMR) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This approach involves redirecting nerves from the residual limb to specific muscles, while disconnecting their original neural connections. TMR not only restores motor signals from the residual nerve but also facilitates the collection of EMG signals from the target muscle. This enables regenerated nerve axons to produce action potentials\u0026mdash;electrical signals that activate muscles in the residual limb, thereby restoring function [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Subsequently, limb muscles serve as a pathway for nerve signals to reach the skin surface, generating new EMG signals that can enhance prosthetic control [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. TMR technology holds promise for improving EMG prosthetic functionality by potentially rehabilitating joint movements and enabling intricate multi-joint motions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, TMR shows potential in restoring movement capabilities and alleviating discomfort in lower limb amputees, fostering optimism for fully functional prosthetic limbs in the future [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCompared to the well-established TMR technique, Targeted Nerve Function Replacement (TNFR) offers a novel approach by directly reconnecting the target nerve to its original neural pathway, which may lead to superior outcomes in preserving motor and sensory functions while minimizing denervation-induced atrophy. However, TMR surgery involves severing the original neural connections of the target muscle, which can disrupt the normal activity of proteins and metabolic enzymes within those muscles. Additionally, the severed nerve endings undergo Wallerian degeneration, gradually breaking down over time due to a lack of stimulation from the neuron cell body. This degeneration impairs the ability of regenerated axons or nerve fibers to effectively communicate with distant denervated skeletal muscles, exacerbating muscle weakness and atrophy. During this phase, skeletal muscle volume decreases, leading to a significant reduction or complete loss of contraction function. Muscle fibers may develop severe fibrosis and risk irreversible atrophy, ultimately resulting in the loss of motor and sensory functions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To address these challenges, researchers have developed several innovative techniques for nerve repair and reinnervation. It is important to distinguish between three key approaches that have emerged in this field: TMR, Regenerative Peripheral Nerve Interfaces (RPNI), and TNFR. TMR, as previously described, redirects severed nerves from the amputated limb to new target muscles after deliberately denervating those muscles. While effective for prosthetic control, this approach sacrifices the original innervation of the target muscles, potentially leading to complications associated with denervation. RPNI employs a different strategy, utilizing free muscle grafts that are carefully wrapped around the terminal ends of transected peripheral nerves [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These grafts function as biological amplifiers for nerve signals, providing stable interfaces between peripheral nerves and artificial limbs without necessitating the denervation of existing functional muscles. TNFR, the central focus of this study, represents a fundamentally distinct approach. Rather than redirecting nerves to new muscles (as in TMR) or using muscle grafts (as in RPNI), TNFR establishes a direct end-to-end anastomosis between an intact donor nerve and the original nerve of a target muscle. This sophisticated technique preserves existing neural pathways while providing supplementary neural input to the target muscle.\u003c/p\u003e \u003cp\u003eIn our precisely engineered TNFR model, we perform a meticulous microsurgical procedure where the median nerve serves as the donor nerve and connects to the musculocutaneous nerve that innervates the brachialis muscle. The anastomosis is strategically positioned at the nerve entry point to the target muscle, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This critical positioning protects motor neurons from prolonged denervation by maintaining neural connections to the target muscle while redirecting potential damage away from the proximal regions containing vital motor neuron bodies toward more distal nerve segments.\u003c/p\u003e \u003cp\u003eThe TNFR procedure begins with careful exposure of both the donor nerve (median nerve) and the recipient nerve (musculocutaneous nerve) near its entry point to the brachialis muscle. The recipient nerve undergoes proximal transection, while the donor nerve is partially transected to create a specialized branch for anastomosis, thereby preserving a portion of its original function. This strategic partial transection creates an effective neural pathway that enables signals from the donor nerve to reach the denervated muscle, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. TNFR preserves muscle viability and function after significant injury by maintaining some original nerve function while adding new neural input. The timing of TNFR intervention likely affects recovery outcomes, much like the established \"golden period\" for nerve repair [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although this concept is widely discussed, there remains ongoing debate regarding its exact timeframe, suggesting that in clinical practice, the timing of nerve repair may need to be customized based on individual circumstances. Despite recent advances, the specific impact of TNFR surgery timing on functional outcomes remains incompletely understood, necessitating further research to determine optimal intervention windows for maximizing recovery potential. TNFR involves creating an anastomosis between the target nerve and the original nerve of the target muscle, rather than simply implanting the target nerve into the muscle. This method facilitates reinnervation of the target muscle by its original nerve, thereby restoring both motor and sensory functions. Additionally, precise and consistent surgical incisions during nerve repair procedures generally promote faster wound healing and better functional outcomes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While studies on related techniques such as RPNI have demonstrated benefits of careful surgical approach [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. TNFR's unique methodology\u0026ndash;connecting an intact donor nerve to the recipient nerve near the muscle entry point\u0026ndash;warrants specific investigation of its wound healing dynamics. The distinct neural network structure within targeted muscles may enhance reinnervation rates following TNFR. However, various clinical conditions can delay nerve repair interventions, potentially compromising outcomes if the optimal 'golden period' for neural regeneration is missed.This strategic positioning near the muscle entry point helps maintain the nerve's connection to its target muscle, thereby shifting the focus of potential damage to the more distal segments of the nerve rather than the proximal regions containing essential motor neuron structures. This adjustment extends the critical window for effective nerve repair and potentially improves the likelihood of successful reinnervation and functional recovery.\u003c/p\u003e \u003cp\u003eThis study aimed to comprehensively evaluate the recovery of sensory and motor functions in rats that underwent TNFR surgery at different time points after injury. We established a standardized TNFR model using the median nerve for reinnervation and the musculocutaneous nerve innervating the brachialis muscle for anastomosis. Following surgery, we conducted a multifaceted assessment of recovery through intramuscular EMG signal analysis, precise muscle tension measurements, detailed behavioral assessments, quantitative sensory neuron counts in dorsal root ganglia (DRG), and comprehensive motor neuron evaluation in the spinal cord.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Animals and experimental design\u003c/h2\u003e\n \u003cp\u003eThe selection of Sprague-Dawley rats was based on their extensive use in nerve injury models, which provides a consistent framework for evaluating TNFR interventions. Although this strain is prone to autotomy behaviors post-injury, their high availability and well-characterized physiology make them a preferred model for initial exploratory studies. To address autotomy-related limitations, postoperative care protocols were strictly implemented. Additionally, the experimental design aimed to minimize variability by using age-matched and weight-matched subjects under controlled environmental conditions. Thirty Specific Pathogen-Free (SPF) adult male Sprague-Dawley rats, aged 7\u0026ndash;8 weeks with a body weight of 220\u0026ndash;250 g, were purchased from the Guangdong Medical Laboratory Animal Center, Guangzhou, China (license No. SCXK (Yue) 2013-0002). The rats were kept in an SPF environment at the Zhuhai campus of Zunyi Medical University, Zhuhai, China.The rats were housed in cages (n\u0026thinsp;=\u0026thinsp;3/cage) under controlled conditions, with a temperature of 22\u0026ndash;26 ℃, relative humidity of 40\u0026ndash;60%, and a 12:12 h light-dark cycle, with free access to food and water. The rats were randomly divided into five groups: control, denervated, TNFR, 2-week delayed TNFR (TNFR-2W), and 4-week delayed TNFR (TNFR-4W), with six rats in each group. The study protocol was approved by the Animal Ethics Committee of the Zhuhai campus of Zunyi Medical University, Zhuhai, China (approval No. 2019-2-273) on March 11, 2019. The study was conducted in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.1 Experimental groups and timeline\u003c/h2\u003e\n \u003cp\u003eThe experiment was designed to evaluate the effectiveness of TNFR at different time points following denervation. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the experimental timeline for all groups. After acclimatization, all experimental groups except the Control underwent median nerve transection on Day 0. The TNFR group received the TNFR procedure immediately after nerve transection. The TNFR-2W group received delayed TNFR surgery 2 weeks after nerve transection (Day 14), while the TNFR-4W group received delayed TNFR surgery 4 weeks after nerve transection (Day 28). Functional and histological evaluations were conducted at 4 weeks post-TNFR surgery for all groups.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Surgical procedures\u003c/h2\u003e\n \u003cp\u003eA TNFR model was established following the TMR surgery protocol described by Jianping Huang et al. [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Rats were deprived of food for 12 hours and water for 4 hours prior to surgery. This fasting protocol was implemented to minimize the risk of aspiration during anesthesia, a standard precaution in rodent surgical procedures. Perioperative analgesia was administered to ensure animal welfare. Buprenorphine (0.05 mg/kg) was given subcutaneously 30 minutes prior to surgery to manage pain and provide prolonged analgesic effects. Additional doses were administered every 12 hours postoperatively for 48 hours to ensure adequate pain relief during the recovery period. Anesthesia was induced with 3% isoflurane for approximately 3\u0026ndash;5 minutes and maintained at 2% isoflurane throughout the operation. The anesthetized rats were fixed in a supine position on the operating table, and the surgical area was fully exposed and depilated for skin preparation. The right ventral side of each group of rats was used as the experimental side. The right median nerve was anastomosed with the musculocutaneous nerve, and recording electrodes were implanted in the biceps brachii. The left ventral side was used as the normal side, only the recording electrodes were implanted, and the rest was left untreated.The area was disinfected with povidone-iodine and then deiodinated with 75% alcohol after drying. A surgical incision was made 1.5-2 cm perpendicular to the midline of the elbow from the acromion. The skin, superficial fascia (subcutaneous tissue), and deep fascia were sequentially opened to isolate the musculocutaneous nerve and the connective tissue adjacent to the median nerve. Once the nerve was fully exposed, the proximal end of the musculocutaneous nerve and the distal end of the median nerve were ligated with an 8\u0026thinsp;\u0026minus;\u0026thinsp;0 suture and severed. An end-to-end anastomosis was performed as close as possible to the point where the biceps nerve enters the muscleFollowing nerve anastomosis, we disinfected and sutured the surgical area in layers (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Meanwhile, in the control group, only the nerve was exposed, and in the denervated group, only denervation (without anastomosis) was conducted, while in the TNFR-2W group, anastomosis was conducted 2 weeks post-denervation, and in the TNFR-4W group, it was conducted 4 weeks post-denervation.\u003c/p\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1 Implantation of EMG Electrodes in Rats\u003c/h2\u003e\n \u003cp\u003eWe surgically implanted EMG electrodes into the rats\u0026apos; bilateral biceps brachii muscles. Each rat was positioned prone and secured using a stereotactic device, with an incision made to expose the skull and remove the fascia. A stereotactic device was then implanted and anchored in the skull of each rat to ensure precise and stable placement of electrodes for chronic neural and muscle activity recordings. This setup allows for accurate targeting of specific anatomical sites and minimizes movement-related artifacts, which is essential for obtaining reliable, consistent data across experimental sessions. By securing the device within the skull, we prevented any shifting or dislodgement, which could compromise data quality and animal safety.\u003c/p\u003e\n \u003cp\u003eA precise 1 mm hole was drilled near the \u0026apos;person\u0026apos; seam, into which a skull nail was inserted, followed by a five-channel connector pre-welded to Teflon-coated stainless steel electrode wires. This assembly was affixed to the skull using ultraviolet-curing adhesive (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Subsequently, a transverse incision above the biceps brachii muscles facilitated access, allowing for careful dissection and exposure of the muscle bellies down to the elbow joint. Surgical forceps were used to subcutaneously route the electrode wires from the skull joint to each biceps brachii muscle, with approximately 5 mm of Teflon coating stripped from the wire ends before meticulous insertion into the muscle bellies. The electrodes were secured using a No. 8 suture needle, and the procedure was concluded with suturing of the incisions and administration of penicillin for anti-inflammatory purposes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e2.3 EMG Signal Acquisition and Recording\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003cp\u003eEMG signals play a crucial role in evaluating surgical outcomes and assessing functional recovery. Following established methodologies for intramuscular EMG recording and analysis [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e], intramuscular EMG signals were recorded using bipolar configuration. Our approach incorporated techniques similar to those described by Boeltz et al. [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] for recording signals from reinnervated muscles, with signal processing methods adapted from studies on EMG analysis in neuromuscular assessment [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. A five-channel connector (Omnetic, Minneapolis, MN, USA) was fixed on the skull with skull nails. Teflon-coated stainless steel wires (Cat# 793500, A-M System Inc, Sequim, WA, USA) were passed subcutaneously to the back (grounded electrode) and both biceps brachii (recording electrodes). After exposing the muscles bilaterally through transverse skin incisions, a 3 mm notch was made in the Teflon coating of the electrodes before implantation. Intramuscular myoelectric signals were collected for 4 weeks starting 1 week after the experimental operation. The electrical stimulation was performed using an invasive approach, with direct surgical exposure of the target median nerve. The EMG signal data were acquired using a self-developed multichannel biopotential signal system at a sampling rate of 1024 Hz (NES-128B01, 128 channels, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China). During the EMG recording, rats performed locomotion on a self-developed wheel treadmill (diameter, 0.4 m; circumference, 1.26 m; maximum speed of 30 r/min [0.63 m/s]). For formal experiments, the parameters were set as follows: running time, 30 seconds; rest time, 30 seconds; number of cycles, 3 (total time, 3 minutes); and speed, 9 r/min (0.19 m/s) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 EMG Signal Processing and Analysis\u003c/h2\u003e\n \u003cp\u003eThe analysis methods were designed to comprehensively assess the intensity, power, and frequency characteristics of EMG signals. First, raw EMG signals were preprocessed by applying a 4th-order Butterworth bandpass filter (20\u0026ndash;150 Hz) directly in the time domain to remove motion artifacts and high-frequency noise while preserving physiologically relevant components. This approach was selected over frequency domain filtering as it is more appropriate for real-time bioelectrical signal processing and has become standard practice in EMG analysis. A dual approach combining Short-Time Fourier Transform (STFT) for time-frequency analysis and Root Mean Square (RMS) value calculations was implemented. This approach captures frequency variations over time, providing insights into the dynamic nature of muscle activity post-surgery. The amplitude was derived as the modulus (absolute value) of the STFT results:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003eThe parameters for the Short-Time Fourier Transform (STFT) were chosen thoughtfully to enhance the analysis of the signal characteristics. The window function employed was the Hamming window, chosen for its optimal trade-off between main-lobe width (3.3 dB) and side-lobe suppression (-42 dB), minimizing spectral leakage compared to rectangular windows. Window length was set to 256 samples (250 ms at 1024 Hz sampling rate). This duration captures 5 cycles of the lowest frequency component (20 Hz) while maintaining quasi-stationarity within each window. Overlap of 50% (128 samples) was implemented to reduce edge effects and improve temporal resolution to 125 ms. Additionally, zero-padding to 512 points enhanced the frequency resolution to 2 Hz. The STFT was computed using MATLAB\u0026apos;s spectrogram function (Signal Processing Toolbox, R2020a) with the aforementioned parameters. The RMS value was utilized to quantify the total energy of the EMG)signal, integrating both amplitude and contraction duration:\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:\\text{RMS}\\text{}=\\sqrt{\\frac{1}{N}\\sum\\:_{i=1}^{N}\\left({x}_{i}^{2}\\right)}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere N represents the total number of samples in each 1-second window (1024 samples at our sampling rate), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:x\\_i\\)\u003c/span\u003e\u003c/span\u003e represents the amplitude value of the EMG signal at sample i (unit: \u0026micro;V).\u003c/p\u003e\n \u003cp\u003eFor all analyses, we specifically selected the central 20-second period of EMG activity from each 30-second running period to avoid transitional effects at the beginning and end of each running cycle. Within this 20-second stable activity period, RMS values were calculated from consecutive 1-second segments with 50% overlap (0.5-second step size), resulting in 39 overlapping windows for each 20-second period. For each rat, we computed the mean of these RMS values across all analyzed segments within each recording session, providing a single representative measure of muscle activity intensity for statistical comparison between experimental groups. Additionally, we calculated the standard deviation of RMS values to assess the variability of muscle activity within each session. The frequency band of 40\u0026ndash;80 Hz was specifically monitored in the STFT results, as this mid-frequency range has been shown to be most sensitive to changes in motor unit recruitment patterns during recovery [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. All signal processing and analyses were performed using MATLAB software (Matlab R2020, MathWorks, Natick, MA, USA). In Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, the x-axis represents time (milliseconds) and the y-axis represents amplitude (microvolts). The combination of amplitude and RMS provides a balanced assessment of muscle activity, capturing both the magnitude and power of the EMG signals. This dual approach ensures a thorough evaluation of muscle function under different experimental conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Measurement of behavioral function\u003c/h2\u003e\n \u003cp\u003eThe post-surgery recovery of phantom limb pain in rats was observed through their autophagy behavior. After collecting intramuscular EMG data, we observed the rats daily for self-injurious behavior. The degree of autophagy in toes is evaluated using a scoring system where a maximum of 13 points can be assigned [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Autophagy Scoring System\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003eOne point is awarded for damage to one or more toenails; one point is given for damage to the distal segment of each toe, with each affected toe contributing one point; if autophagy occurs in the distal segment of all toes, one point is awarded in total regardless of the number of toes affected; one point is given for damage to the proximal phalangeal segment of each toe, with each affected toe contributing one point; and if autophagy occurs in the proximal phalangeal segment of all toes, one point is awarded in total regardless of the number of toes affected. This system ensures consistent point allocation based on the specific damage or autophagy observed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 In vivo mechanical analysis of biceps brachii\u003c/h2\u003e\n \u003cp\u003eThe in vivo mechanical analysis of the biceps brachii muscle provides valuable insights into its functionality under both normal and pathological conditions, and helps in assessing the efficacy of various treatments, including post-surgical recovery [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Following the observation of autophagic behaviors, we conducted an examination of the biomechanical properties of the biceps brachii muscle using the Melab-u/8c502 biosignal acquisition system, equipped with a pressure sensor capable of handling up to 50 grams (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). To ensure accurate measurements, we stimulated the proximal region of the nerve graft. This allowed us to evaluate the functional mimicry between the median nerve and the musculocutaneous nerve subsequent to surgical interventions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Hematoxylin and eosin (H.E.) staining of the DRG\u003c/h2\u003e\n \u003cp\u003eAfter measuring muscle strength, morphological observations were conducted using Hematoxylin and Eosin (H.E.) staining to examine the dorsal root ganglia (DRG), which are critical structures where sensory neurons converge. This technique allowed us to identify and assess the characteristics of these neurons within the ganglia. Following the in vivo mechanical analysis, the rats were anesthetized with isoflurane and cannulated through the left ventricle to the ascending aorta for perfusion fixation. The spinal cord and DRG of the C5-T1 segment were removed and immediately fixed in 4% paraformaldehyde for 24 hours. The samples were dehydrated using a gradient sucrose solution, embedded in an optimal cutting temperature compound, and stored at -80 ℃. The embedded DRG samples were then sectioned at 20\u0026micro;m thickness using a Leica CM1950 cryostat, and stained with H.E. stain (ZSGBBio, Beijing, China). The slides were observed under an upright microscope (Eclipse E100, Nikon, Japan) with a 40\u0026times; objective lens, and the number of neurons was counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Immunofluorescence staining of the spinal cord\u003c/h2\u003e\n \u003cp\u003eFinally, the spinal cord was subjected to immunofluorescence staining to assess the distribution and morphology of synapses on motor neurons. Synaptophysin (SYN) and postsynaptic density protein 95 (PSD-95) are pivotal proteins that modulate neuromuscular junction function and play crucial roles in nerve signal transmission and muscle action [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Spinal cord tissues embedded in paraffin were sectioned at 15\u0026ndash;20 \u0026micro;m thickness using a Leica cryostat at -20 ℃. These sections were then deparaffinized, rehydrated, and subjected to antigen retrieval.\u003c/p\u003e\n \u003cp\u003eThe sections were blocked with bovine serum albumin and incubated with primary antibodies against SYN (rabbit anti-SYN antibody, 1:500, Cat# bs-23504R, RRID: AB_2895150, Bioss) and PSD-95 (mouse-produced primary antibody, specific catalog and concentration to be added). Following primary antibody incubation, the sections were incubated with fluorescently labeled secondary antibodies: goat anti-rabbit IgG/Cy3 for SYN (green fluorescence) and goat anti-mouse IgG conjugated to Alexa Fluor 594 for PSD-95 (red fluorescence). After nuclear counterstaining with 4\u0026rsquo;, 6-diamidino-2-phenylindole (DAPI, Abcam), the sections were mounted and observed under an upright fluorescence microscope (E100, Nikon) using a 40x objective lens. The mean optical density of SYN and PSD-95 was quantified using ImageJ software (National Institutes of Health).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eFunctional and histopathological analyses were performed under blinding protocols to ensure unbiased results. The normality test was performed using the Shapiro-Wilk test with a significance level of 0.05. Homogeneity of variances was confirmed with Levene\u0026apos;s test. All data samples conformed to normal distributions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). We present quantitative data as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Differences between experimental groups were assessed using one-way ANOVA, followed by post-hoc Bonferroni tests for all multiple comparisons. This approach was selected to control for Type I error rates across all analyses. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were conducted under blinding protocols using GraphPad Prism software version 9.0.0 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.1 General conditions of rats\u003c/h2\u003e\n \u003cp\u003ePost-surgery, all rats exhibited normal feeding behavior and showed no signs of infections, ulcers, or nervous system disorders.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.2 Effect of TNFR surgery on intramuscular EMG signal analysis in rats\u003c/h2\u003e\n \u003cp\u003eEMG signals were successfully collected from rats 4 weeks post-surgery. Figures 7A-E and 8A-E show the typical averaged waveforms and muscle activity spectra of EMG signals from all groups (control, denervation, TNFR, TNFR-2W, and TNFR-4W), respectively. The biceps brachii muscles of the TNFR, TNFR-2W, and TNFR-4W groups produced EMG signals of varying amplitudes post-TNFR surgery (Figs. 7B-D and 8B-D). The amplitude and muscle activity of EMG signals in the TNFR group were significantly higher than those observed in all other experimental groups. In stark contrast, the denervated group exhibited only background noise with no detectable EMG signals (Figs. 7E and 8E), confirming complete loss of neuromuscular activity. The comprehensive statistical analysis of intramuscular signal parameters across all experimental conditions is presented in Figs. 7F and 8F, which display the quantitative amplitude and RMS values, respectively. These analyses clearly demonstrate the superior functional recovery achieved with immediate TNFR intervention compared to delayed procedures.\u003c/p\u003e\n \u003cp\u003eSignificant differences were observed in the amplitude and RMS values among the groups (Figs. 7F and 8F). The amplitude and RMS values were significantly higher in the control group compared to the TNFR group (P \u0026lt; 0.05). Within the TNFR groups, the amplitude and RMS values were higher in the TNFR group than in the TNFR-2W and TNFR-4W groups (P \u0026lt; 0.05). Amplitude and RMS values were significantly lower in the denervation group compared to the other groups (P \u0026lt; 0.05). While there was no significant difference in amplitude and RMS values between the TNFR-2W and TNFR-4W groups (P \u0026gt; 0.05), both groups exhibited amplitude and RMS values significantly higher than those of the denervation group (P \u0026lt; 0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e3.3 Autotomy behavior observation\u003c/h2\u003e\n \u003cp\u003eThe experimental results indicated that the control and TNFR groups displayed no abnormalities in toe morphology. However, in the denervation group, phantom limb pain and abnormal autophagy behavior were observed as early as one week post-surgery, with symptoms intensifying over time (Fig. 9). The TNFR-2W and TNFR-4W groups exhibited autophagy, redness, and swelling of the toes at one week post-surgery, but these symptoms, along with the phantom limb pain, decreased and eventually subsided within 14–21 days. Table 2 shows the autotomy behavior in rats across different time points for each experimental group. In the TNFR-2W group, 3 rats exhibited level 1 autotomy behavior in Week 1 while 3 had no symptoms; 4 rats showed level 2 behavior in Week 2 with 2 remaining asymptomatic; by Week 3, only 2 rats still displayed level 1 behavior with 4 recovered to no symptoms; and by Week 4, all 6 rats had completely recovered with no symptoms. The TNFR-4W group showed a similar recovery pattern, with 3 rats displaying level 1 behavior in Week 1, progressing to 4 rats with level 2 behavior in Week 2, then improving to 4 rats with level 1 behavior in Week 3, and finally all 6 rats showing complete recovery by Week 4. In contrast, rats in the denervation group demonstrated progressively worsening symptoms, with 5 rats showing level 1 autotomy in Week 1, all 6 rats reaching level 2 in Week 2, and all 6 rats progressing to severe level 3 autotomy behavior by Weeks 3 and 4. This demonstrates that without intervention, denervated rats experienced continual deterioration in symptoms throughout the observation period. Additionally, by the end of the observation period, the toe morphologyin the TNFR intervention groups (TNFR-2W and TNFR-4W) appeared similar to that observed in the control group, suggesting resolution of the initial autotomy symptoms. This demonstrates that without intervention, denervated rats experienced continual deterioration in symptoms throughout the observation period.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAutophagy of rats in each group. The numbers in parentheses represent the number of animals exhibiting autophagy behavior in each group per week.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSymptom Level\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeek1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeek 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeek 4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 1 (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 2 (++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 3 (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNFR group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 1 (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 2 (++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 3 (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNFR-2W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 1 (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 2 (++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 3 (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNFR-4W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 1 (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 2 (++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 3 (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDenervated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 1 (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 2 (++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevel 3 (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eTNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, and TNFR-4W: 4-week delayed TNFR group\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.4 Effect of TNFR surgery on biceps brachii contraction force\u003c/h2\u003e\n \u003cp\u003eFour weeks post-surgery, both the maximum contraction force and the maximum tetanic contraction force of the biceps brachii were significantly higher in the control group compared to the denervation, TNFR, TNFR-2W, and TNFR-4W groups (P \u0026lt; 0.05) (Fig. 10). Furthermore, the TNFR group showed significantly greater muscle strength than the TNFR-2W, TNFR-4W, and denervation groups (P \u0026lt; 0.05). However, there was no statistically significant difference in muscle strength between the TNFR-2W and TNFR-4W groups (P \u0026gt; 0.05), though both were significantly stronger than the denervation group (P \u0026lt; 0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.5 Effects of TNFR surgery on the DRG neurons in rats\u003c/h2\u003e\n \u003cp\u003eH.E. staining was performed on the DRG samples of each group. The number of sensory neurons was significantly higher in the TNFR group than in the TNFR-2W and TNFR-4W groups (P \u0026lt; 0.05; Fig. 11). The TNFR-2W group showed a trend toward better preservation of neuronal morphology compared to the TNFR-4W and denervation groups, although this difference was not statistically significant (P \u0026gt; 0.05)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.6 Effects of TNFR surgery on SYN and PSD-95 expression in the spinal cord motor neurons in rats\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eImmunoreactivity in the motor neurons of the anterior horn of the spinal cord in control group rats. Compared with the control group, the SYN immunoreactivity in the TNFR group showed a reduction (P \u0026gt; 0.05; Fig. 12P). The SYN immunoreactivity was significantly higher in the TNFR group compared to the TNFR-2W, TNFR-4W, and denervation groups (P \u0026lt; 0.05; Fig. 12P), demonstrating better preservation of presynaptic terminals with immediate intervention. For PSD-95 expression, all experimental groups (TNFR, TNFR-2W, TNFR-4W, and denervation groups) showed significantly reduced levels compared to the control group (P \u0026lt; 0.05; Fig. 12Q). Importantly, there were no significant differences in PSD-95 immunoreactivity among all experimental groups (P \u0026gt; 0.05; Fig. 12Q), indicating that intervention timing had no differential impact on postsynaptic protein expression. While fluorescence intensity provides valuable quantitative and spatial information on protein expression, it may be influenced by external factors such as staining time and antibody concentration. To mitigate these potential variables, we implemented rigorous methodological controls including standardized staining protocols, consistent antibody lots, uniform incubation times, and calibrated image acquisition settings across all experimental groups. This standardization ensures that the observed differences in SYN and PSD-95 expression patterns (Fig. 12) accurately reflect biological changes rather than technical variations.\u003c/p\u003e\n \u003cp\u003eRepresentative immunofluorescence staining images of SYN/PSD-95 in the control (A), denervated (B), TNFR (C), TNFR-2W (D), and TNFR-4W (E) groups ( scale bar: 200 µm, original magnification 4×). (F-J) Magnified images of the SYN/PSD-95-stained areas in A-E (scale bar: 50 µm, original magnification 10×). (K-O) Merged images showing co-localization. Quantitative analysis of (P) relative fluorescence intensity of SYN and (Q) relative fluorescence intensity of PSD-95 across groups. Data are expressed as mean ± SD, n = 6 per group. Statistical analysis: one-way ANOVA (SYN: F (4, 25) = 18.72, P \u0026lt; 0.0001; PSD-95: F (4, 25) = 31.24, P \u0026lt; 0.0001) followed by Bonferroni multiple comparisons test. *indicates the difference between the control group and other groups, *P \u0026lt; 0.05; #indicates the difference between TNFR and other groups, #P \u0026lt; 0.05. SYN: synaptophysin, PSD-95: post-synaptic density protein 95, TNFR: targeted nerve function replacement, TNFR-2W: 2-week delayed TNFR group, TNFR-4W: 4-week delayed TNFR group, and SD: standard deviation\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this experiment, we demonstrated that immediate nerve reinnervation following injury significantly improves muscle function, showing markedly better results compared to reinnervation delayed by 2 and 4 weeks. Additionally, we found that combining implanted electrodes with TNFR is an effective method for in vivo tracking of functional recovery. Our research confirms the feasibility of functional restoration through TNFR surgery, supported by both bio-structural and EMG data as shown in Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, which is crucial for the development of myoelectric prosthetics. Early detection of signals from the target muscle post-surgery indicates a significant reduction in overall functional recovery time. These findings provide important evidence for further optimizing nerve reinnervation surgery.\u003c/p\u003e\n\u003cp\u003eTMR can significantly enhance axon regeneration, increase the quantity and size of regenerated axons, shorten the duration of muscle reinnervation, and prevent neuromas [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, our TNFR approach offers distinct advantages by maintaining original neural pathways while providing supplementary neural input. In this work, we strategically placed the site for neurorrhaphy close to the target muscle to reduce both the regeneration distance and duration as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. To achieve superior results, we made several refinements to the experimental protocol. Initially, we used a pull hook to elevate the pectoralis major, pectoralis minor, and portions of the deltoid. Although this could have prevented muscle trauma, it did not provide a satisfactory view of the operative site. Therefore, we opted to carefully separate the pectoralis major and minor muscles to obtain a clear view of the operation site, reducing surgical time despite its unfavorable effect on motor function. Regarding the surgical approach, following guidelines from a previous study [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], we ensured optimal conditions for nerve reinnervation. By incising the musculocutaneous nerve (MCN) proximally and the median nerve (MN) distally, we increased nerve length, facilitating a tension-free neurorrhaphy as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC. We refined our surgical procedure to prevent damage to the local vascular network and limit nerve disconnection to only the necessary segments for ligation. This approach effectively reduced bleeding and enhanced blood supply to the transected nerve, crucial for delivering nutrients and regeneration factors [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Neovascularization served as a conduit for the restored nerve, directing regenerating axons [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e] and significantly reducing recovery time. We performed second-stage operations with remarkable precision and ease. The surgical field was unobstructed, allowing for direct and effortless nerve access. This streamlined approach resulted from meticulous planning and execution, ensuring minimal tissue manipulation to effectively expose target areas. Consequently, this may have contributed to reduced fibroblast infiltration and lower damage to blood vessels.\u003c/p\u003e\n\u003cp\u003eFor functional assessment, the treadmill examination is a streamlined and precise method to regulate and assess the behavioral parameters of rats during training regimens [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this project, we used a customized wheel treadmill as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, unlike conventional flat treadmills, to enhance engagement and activity in the forelimbs. Our preliminary tests revealed that the wheel treadmill prevented the rats from leaping forward using their hind limbs, thereby compelling increased reliance on and participation of the forelimbs. After a brief training period, the rats demonstrated improved balance and active participation on the wheel treadmill. This approach provided an effective strategy for observing and assessing the recovery trajectory of forelimb muscle functionality and neural control.\u003c/p\u003e\n\u003cp\u003eTo validate this method, we also measured the maximum contraction force and maximum tetanic contraction force of the biceps brachii muscle presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA-B, which helped assess the function and health status of the muscle fibers. In the TNFR group, the immediate application of TNFR following an injury resulted in the highest myoelectric signal amplitudes, maximum contraction force, and maximum tetanic contraction force compared with the other experimental groups. In contrast, both signal amplitude and muscle power significantly decreased in the delayed TNFR groups, specifically the TNFR-2W and TNFR-4W groups. Therefore, immediate TNFR preserved significantly better muscle function compared with delayed intervention.\u003c/p\u003e\n\u003cp\u003eIrreversible muscle atrophy post-denervation, as described by Soendenbroe [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], can lead to severe muscle weakness and unfavorable functional prognoses. This accounts for the poor performance observed in the denervation group during functional assessments, where all three evaluation indices were lower than those of the other groups as demonstrated in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eF, and \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA-B. The delayed TNFR groups showed no significant differences in their myoelectric signal patterns and muscle power, consistent with the results of the functional assessment. These results indicate the significant benefits of TNFR, especially immediate TNFR intervention, for effective restoration of motor function.\u003c/p\u003e\n\u003cp\u003eOur findings on the timing-dependent efficacy of nerve reinnervation parallel recent evidence in related fields. A review by Dominguez et al. [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e] demonstrated similar timing-dependent effects with TMR, where acute TMR showed superior pain management outcomes compared to delayed procedures, further supporting the critical importance of early intervention in neural pathway restoration.\u003c/p\u003e\n\u003cp\u003eRegarding behavioral outcomes, while phantom limb pain typically occurs in individuals following limb amputation, the observed self-mutilation behaviors (or autotomy) in our study may indicate neuropathic pain or sensory disturbances associated with delayed nerve repair rather than phantom pain. Autotomy behaviors were observed in rats that underwent delayed surgeries, which involved nerve severance, while those receiving immediate surgery did not exhibit these signs as documented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Autotomy behaviors rarely follow median nerve injury [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. We selected Sprague Dawley rats for their availability and established use in nerve injury models. However, Carr et al. [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], noted this strain shows higher rates of autotomy after injury compared to Lewis rats, which exhibit much lower rates of this behavior. Future studies might consider using alternative strains with lower susceptibility to autotomy to further minimize these effects and clarify if the autophagy behavior observed is strain-specific or model-dependent. These results suggest that immediate TNFR intervention yields the best results in nerve reconstruction.\u003c/p\u003e\n\u003cp\u003eFor histological findings, DRG are composed of sensory fiber cells that receive all nerve impulses, including general somatosensory and visceral sensations, from the body\u0026apos;s receptors. These impulses are then relayed to the spinal cord via sensory fibers. In this study, we used H.E. staining to examine the morphology and number of sensory neurons in the DRG as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA-O. Our analysis revealed that TNFR intervention effectively normalized neuronal morphology, restoring the size of the cell bodies and the number of axons to levels statistically similar to those in the control group. However, delayed TNFR treatment in the TNFR-2W and TNFR-4W groups only partially recovered sensory neuron morphology and functionality, with observed instances of demyelination. Quantitative analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eP) confirmed a significant reduction in sensory neuron numbers in the TNFR-2W and TNFR-4W groups compared to immediate TNFR. Therefore, timely TNFR intervention, particularly when implemented immediately, is essential for the effective recovery of sensory neurons in the DRG.\u003c/p\u003e\n\u003cp\u003eRegarding synaptic marker analysis, SYN protein is localized to synaptic vesicles in the presynaptic terminals of neurons. It is involved in the release of activity-dependent neurotransmitters and plays a crucial role in synaptic plasticity. In contrast, PSD-95 is essential for post-synaptic signal transduction and synaptic plasticity by anchoring receptor proteins at the synaptic membrane [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e] Alterations in PSD-95 expression in motor neurons can affect synaptic strength, contributing to changes in motor activity.\u003c/p\u003e\n\u003cp\u003eOur results showed a significant decrease in the expression of SYN and PSD-95 across all experimental groups compared to the control group as visualized in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA-O. SYN, a presynaptic vesicle protein essential for neurotransmitter release, showed relatively better restoration in the immediate TNFR group compared to the TNFR-2W, TNFR-4W, and denervated groups.\u003c/p\u003e\n\u003cp\u003eHowever, PSD-95, a scaffolding protein critical for postsynaptic organization and signaling, remained similarly reduced across all intervention groups as quantified in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eP-Q, suggesting differential effects on pre-and postsynaptic markers.\u003c/p\u003e\n\u003cp\u003eThis pattern indicates that while immediate TNFR intervention may partially preserve presynaptic structures (as evidenced by improved SYN expression), postsynaptic architecture (represented by PSD-95) appears more resistant to recovery regardless of intervention timing. The persistent reduction in PSD-95 across all experimental groups suggests that postsynaptic densities in motor neurons within the anterior horn of the spinal cord may require additional interventions beyond TNFR to achieve complete recovery. This differential response between presynaptic and postsynaptic markers highlights the complex nature of synaptic reorganization following peripheral nerve injury and suggests that comprehensive neuronal recovery may require targeted approaches that address both pre- and postsynaptic elements of the neural circuit.\u003c/p\u003e\n"},{"header":"5. Limitations","content":"\u003cp\u003eFirst, our rat model may not fully represent the complexity of human peripheral nerve injuries due to anatomical and physiological differences. Second, while we demonstrated functional recovery through EMG and behavioral assessments, the molecular mechanisms underlying TNFR-mediated neuronal survival remain unexplored. Third, the observed autotomy behavior in Sprague Dawley rats may be strain-specific and should be interpreted cautiously when translating to clinical applications. Finally, challenges in maintaining EMG signal quality over extended periods due to potential immune rejection and electrode degradation warrant future development of more biocompatible electrode arrays to enhance signal fidelity for long-term monitoring.\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eThis study demonstrated that TNFR effectively promotes functional recovery following nerve injury in rats, with immediate intervention yielding significantly better outcomes than delayed procedures. Our comprehensive evaluation revealed superior EMG signals, reduced autophagic behavior, and better preservation of muscle function and neuronal structures with immediate TNFR. These findings highlight the critical importance of early intervention in peripheral nerve injuries and provide valuable insights for improving surgical strategies and neuroprosthetic development in clinical settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA: Analysis of Variance\u003c/p\u003e\n\u003cp\u003eARRIVE: Animal Research: Reporting of In Vivo Experiments\u003c/p\u003e\n\u003cp\u003eDRG: Dorsal Root Ganglion\u003c/p\u003e\n\u003cp\u003eEMG: Electromyography\u003c/p\u003e\n\u003cp\u003eGND: Ground\u003c/p\u003e\n\u003cp\u003eH.E.: Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eMCN: Musculocutaneous Nerve\u003c/p\u003e\n\u003cp\u003eMN: Median Nerve\u003c/p\u003e\n\u003cp\u003ePSD-95: Postsynaptic Density Protein 95\u003c/p\u003e\n\u003cp\u003eRPNI: Regenerative Peripheral Nerve Interfaces\u003c/p\u003e\n\u003cp\u003eRMS: Root Mean Square\u003c/p\u003e\n\u003cp\u003eSPF: Specific Pathogen-Free\u003c/p\u003e\n\u003cp\u003eSTFT: Short-Time Fourier Transform\u003c/p\u003e\n\u003cp\u003eSYN: Synaptophysin\u003c/p\u003e\n\u003cp\u003eTMR: Targeted Muscle Reinnervation\u003c/p\u003e\n\u003cp\u003eTNFR: Targeted Nerve Function Replacement\u003c/p\u003e\n\u003cp\u003eTNFR-2W: 2-Week Delayed Targeted Nerve Function Replacement\u003c/p\u003e\n\u003cp\u003eTNFR-4W: 4-Week Delayed Targeted Nerve Function Replacement\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study conception and design were formulated by Chunxiao Tang and Yuanheng Li. Chunxiao Tang conducted the formal analysis, methodology, investigation, and drafted the original manuscript. Yuanheng Li contributed to the investigation, software, and formal analysis. Jiamei Guo and Xinxian Fan assisted with formal analysis, methodology, and investigation. Yifeng Lin was responsible for producing the article illustrations. Yifan Gao and Lin Yang provided supervision, investigation, and editorial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been supported by grants from \u0026nbsp;National Natural Science Foundation of China (#82260456, 81921804), Science and Technology Planning Project of Shenzhen (JCYJ20230807140559047), Science and Technology Department of Guizhou Province (202342938082710225), Zunyi city science and technology plan project(HZ-2020-56), Zunyi Medical University(F-ZH-015, 2018-5772-063), SIAT College Student Innovation Practice Training Program (2023-38, 2023-20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Animal Ethics Committee of the Zhuhai campus of Zunyi Medical University, Zhuhai, China (approval No. 2019-2-273) on March 11, 2019. The study was conducted in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors consent to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they do not have any competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJensen, S. E., Z. Butt, A. Bill, T. Baker, M. M. Abecassis, A. W. Heinemann, D. Cella, and G. A. Dumanian. \u0026quot;Quality of Life Considerations in Upper Limb Transplantation: Review and Future Directions.\u0026quot; \u003cem\u003eJ Hand Surg Am\u003c/em\u003e 37, no. 10 (2012): 2126-35.\u003c/li\u003e\n\u003cli\u003eKuiken, T. A., L. A. Miller, R. D. Lipschutz, B. A. Lock, K. Stubblefield, P. D. Marasco, P. Zhou, and G. A. Dumanian. \u0026quot;Targeted Reinnervation for Enhanced Prosthetic Arm Function in a Woman with a Proximal Amputation: A Case Study.\u0026quot; \u003cem\u003eLancet\u003c/em\u003e 369, no. 9559 (2007): 371-80.\u003c/li\u003e\n\u003cli\u003eKuiken, Todd A, Guanglin Li, Blair A Lock, Robert D Lipschutz, Laura A Miller, Kathy A Stubblefield, and Kevin B Englehart. \u0026quot;Targeted Muscle Reinnervation for Real-Time Myoelectric Control of Multifunction Artificial Arms.\u0026quot; \u003cem\u003eJama\u003c/em\u003e 301, no. 6 (2009): 619-28.\u003c/li\u003e\n\u003cli\u003eKuiken, Todd A. \u0026quot;The Scientific Basis of Targeted Muscle Reinnervation.\u0026quot; \u003cem\u003eTargeted Muscle Reinnervation: A Neural Interface for Artificial Limbs\u003c/em\u003e 9 (2013).\u003c/li\u003e\n\u003cli\u003eKuiken, T. A., A. K. Barlow, L. Hargrove, and G. A. Dumanian. \u0026quot;Targeted Muscle Reinnervation for the Upper and Lower Extremity.\u0026quot; \u003cem\u003eTech Orthop\u003c/em\u003e 32, no. 2 (2017): 109-16.\u003c/li\u003e\n\u003cli\u003eCheesborough, J. E., L. H. Smith, T. A. Kuiken, and G. A. Dumanian. \u0026quot;Targeted Muscle Reinnervation and Advanced Prosthetic Arms.\u0026quot; \u003cem\u003eSemin Plast Surg\u003c/em\u003e 29, no. 1 (2015): 62-72.\u003c/li\u003e\n\u003cli\u003eVadal\u0026agrave;, G., G. Di Pino, L. Ambrosio, L. Diaz Balzani, and V. Denaro. \u0026quot;Targeted Muscle Reinnervation for Improved Control of Myoelectric Upper Limb Prostheses.\u0026quot; \u003cem\u003eJ Biol Regul Homeost Agents\u003c/em\u003e 31, no. 4 suppl 1 (2017).\u003c/li\u003e\n\u003cli\u003eLuft, M., J. Klepetko, S. Muceli, J. Ib\u0026aacute;\u0026ntilde;ez, V. Tereshenko, C. Festin, G. Laengle, O. Politikou, U. Maierhofer, D. Farina, O. C. Aszmann, and K. D. Bergmeister. \u0026quot;Proof of Concept for Multiple Nerve Transfers to a Single Target Muscle.\u0026quot; \u003cem\u003eElife\u003c/em\u003e 10 (2021).\u003c/li\u003e\n\u003cli\u003eMiller, L. A., R. D. Lipschutz, K. A. Stubblefield, B. A. Lock, H. Huang, T. W. Williams, 3rd, R. F. Weir, and T. A. Kuiken. \u0026quot;Control of a Six Degree of Freedom Prosthetic Arm after Targeted Muscle Reinnervation Surgery.\u0026quot; \u003cem\u003eArch Phys Med Rehabil\u003c/em\u003e 89, no. 11 (2008): 2057-65.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Brien, A. L., S. W. Jordan, J. M. West, L. M. Mioton, G. A. Dumanian, and I. L. Valerio. \u0026quot;Targeted Muscle Reinnervation at the Time of Upper-Extremity Amputation for the Treatment of Pain Severity and Symptoms.\u0026quot; \u003cem\u003eJ Hand Surg Am\u003c/em\u003e 46, no. 1 (2021): 72.e1-72.e10.\u003c/li\u003e\n\u003cli\u003eStoehr, J. R., R. Sood, S. W. Jordan, and G. A. Dumanian. \u0026quot;Targeted Muscle Reinnervation at the Time of Amputation in the Management of Complex Regional Pain Syndrome of the Lower Extremity.\u0026quot; \u003cem\u003eMicrosurgery\u003c/em\u003e 40, no. 8 (2020): 852-58.\u003c/li\u003e\n\u003cli\u003eElAbd, Rawan, Todd Dow, Sinan Jabori, Becher Alhalabi, Samuel J Lin, and Sammy Dowlatshahi. \u0026quot;Pain and Functional Outcomes Following Targeted Muscle Reinnervation: A Systematic Review.\u0026quot; \u003cem\u003ePlastic and Reconstructive Surgery\u003c/em\u003e 153, no. 2 (2024): 494-508.\u003c/li\u003e\n\u003cli\u003eSmith, B. W., A. K. Daunter, L. J. Yang, and T. J. Wilson. \u0026quot;An Update on the Management of Neonatal Brachial Plexus Palsy-Replacing Old Paradigms: A Review.\u0026quot; \u003cem\u003eJAMA Pediatr\u003c/em\u003e 172, no. 6 (2018): 585-91.\u003c/li\u003e\n\u003cli\u003eLeach, G. A., R. A. Dean, N. G. Kumar, C. Tsai, F. E. Chiarappa, P. S. Cederna, T. A. Kung, and C. M. Reid. \u0026quot;Regenerative Peripheral Nerve Interface Surgery: Anatomic and Technical Guide.\u0026quot; \u003cem\u003ePlast Reconstr Surg Glob Open\u003c/em\u003e 11, no. 7 (2023): e5127.\u003c/li\u003e\n\u003cli\u003eFrost, Christopher M., Daniel C. Ursu, Shane M. Flattery, Andrej Nedic, Cheryl A. Hassett, Jana D. Moon, Patrick J. Buchanan, R. Brent Gillespie, Theodore A. Kung, Stephen W. P. Kemp, Paul S. Cederna, and Melanie G. Urbanchek. \u0026quot;Regenerative Peripheral Nerve Interfaces for Real-Time, Proportional Control of a Neuroprosthetic Hand.\u0026quot; \u003cem\u003eJournal of NeuroEngineering and Rehabilitation\u003c/em\u003e 15, no. 1 (2018): 108.\u003c/li\u003e\n\u003cli\u003eWu, Peng. \u0026quot;Delayed Repair of the Peripheral Nerve: A Novel Model in the Rat Sciatic Nerve.\u0026quot; \u003cem\u003eJournal of neuroscience methods\u003c/em\u003e 214, no. 1 (2013): 37-44.\u003c/li\u003e\n\u003cli\u003eXu, C., Y. Kou, P. Zhang, N. Han, X. Yin, J. Deng, B. Chen, and B. Jiang. \u0026quot;Electrical Stimulation Promotes Regeneration of Defective Peripheral Nerves after Delayed Repair Intervals Lasting under One Month.\u0026quot; \u003cem\u003ePLoS One\u003c/em\u003e 9, no. 9 (2014): e105045.\u003c/li\u003e\n\u003cli\u003eGriffin, J. W., M. V. Hogan, A. B. Chhabra, and D. N. Deal. \u0026quot;Peripheral Nerve Repair and Reconstruction.\u0026quot; \u003cem\u003eJ Bone Joint Surg Am\u003c/em\u003e 95, no. 23 (2013): 2144-51.\u003c/li\u003e\n\u003cli\u003eGrinsell, D., and C. P. Keating. \u0026quot;Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies.\u0026quot; \u003cem\u003eBiomed Res Int\u003c/em\u003e 2014 (2014): 698256.\u003c/li\u003e\n\u003cli\u003eKubiak, C. A., S. W. P. Kemp, and P. S. Cederna. \u0026quot;Regenerative Peripheral Nerve Interface for Management of Postamputation Neuroma.\u0026quot; \u003cem\u003eJAMA Surg\u003c/em\u003e 153, no. 7 (2018): 681-82.\u003c/li\u003e\n\u003cli\u003ePercie du Sert, Nathalie, Viki Hurst, Amrita Ahluwalia, Sabina Alam, Marc T. Avey, Monya Baker, William J. Browne, Alejandra Clark, Innes C. Cuthill, Ulrich Dirnagl, Michael Emerson, Paul Garner, Stephen T. Holgate, David W. Howells, Natasha A. Karp, Stanley E. Lazic, Katie Lidster, Catriona J. MacCallum, Malcolm Macleod, Esther J. Pearl, Ole H. Petersen, Frances Rawle, Penny Reynolds, Kieron Rooney, Emily S. Sena, Shai D. Silberberg, Thomas Steckler, and Hanno W\u0026uuml;rbel. \u0026quot;The Arrive Guidelines 2.0: Updated Guidelines for Reporting Animal Research.\u0026quot; \u003cem\u003eBMC Veterinary Research\u003c/em\u003e 16, no. 1 (2020): 242.\u003c/li\u003e\n\u003cli\u003eJianping, Huang, L. I. Wenqing, Yang Lin, Z. H. U. Mingxing, Z. H. U. Xiaodi, L. I. Chuyan, Yang Zijian, and L. I. Guanglin. \u0026quot;A Pilot Study of Nerve Function Reinnervation on a Transhumeral Amputee.\u0026quot; \u003cem\u003eJournal of Integration Technology\u003c/em\u003e 5, no. 5: 30-37.\u003c/li\u003e\n\u003cli\u003eCiti, L., J. Carpaneto, K. Yoshida, K. P. Hoffmann, K. P. Koch, P. Dario, and S. Micera. \u0026quot;On the Use of Wavelet Denoising and Spike Sorting Techniques to Process Electroneurographic Signals Recorded Using Intraneural Electrodes.\u0026quot; \u003cem\u003eJ Neurosci Methods\u003c/em\u003e 172, no. 2 (2008): 294-302.\u003c/li\u003e\n\u003cli\u003eZhong, S., Z. Zhang, H. Su, C. Li, Y. Lin, W. Lu, Z. Jiang, and L. Yang. \u0026quot;Efficacy of Biological and Physical Enhancement on Targeted Muscle Reinnervation.\u0026quot; \u003cem\u003eCyborg Bionic Syst\u003c/em\u003e 2022 (2022): 9759265.\u003c/li\u003e\n\u003cli\u003eBoeltz, T., M. Ireland, K. Mathis, J. Nicolini, K. Poplavski, S. J. Rose, E. Wilson, and A. W. English. \u0026quot;Effects of Treadmill Training on Functional Recovery Following Peripheral Nerve Injury in Rats.\u0026quot; \u003cem\u003eJ Neurophysiol\u003c/em\u003e 109, no. 11 (2013): 2645-57.\u003c/li\u003e\n\u003cli\u003eGriffin, D., and Lim Jae. \u0026quot;Signal Estimation from Modified Short-Time Fourier Transform.\u0026quot; \u003cem\u003eIEEE Transactions on Acoustics, Speech, and Signal Processing\u003c/em\u003e 32, no. 2 (1984): 236-43.\u003c/li\u003e\n\u003cli\u003eCosta, M. V., L. A. Pereira, R. S. Oliveira, R. E. Pedro, T. V. Camata, T. Abrao, M. A. Brunetto, and L. R. Altimari. \u0026quot;Fourier and Wavelet Spectral Analysis of Emg Signals in Maximal Constant Load Dynamic Exercise.\u0026quot; \u003cem\u003eAnnu Int Conf IEEE Eng Med Biol Soc\u003c/em\u003e 2010 (2010): 4622-5.\u003c/li\u003e\n\u003cli\u003eda Silva, R. A., C. Larivi\u0026egrave;re, A. B. Arsenault, S. Nadeau, and A. Plamondon. \u0026quot;The Comparison of Wavelet- and Fourier-Based Electromyographic Indices of Back Muscle Fatigue During Dynamic Contractions: Validity and Reliability Results.\u0026quot; \u003cem\u003eElectromyogr Clin Neurophysiol\u003c/em\u003e 48, no. 3-4 (2008): 147-62.\u003c/li\u003e\n\u003cli\u003eWakeling, J. M., S. A. Pascual, B. M. Nigg, and V. von Tscharner. \u0026quot;Surface Emg Shows Distinct Populations of Muscle Activity When Measured During Sustained Sub-Maximal Exercise.\u0026quot; \u003cem\u003eEur J Appl Physiol\u003c/em\u003e 86, no. 1 (2001): 40-7.\u003c/li\u003e\n\u003cli\u003eHuang, H. J., and D. P. Ferris. \u0026quot;Neural Coupling between Upper and Lower Limbs During Recumbent Stepping.\u0026quot; \u003cem\u003eJ Appl Physiol (1985)\u003c/em\u003e 97, no. 4 (2004): 1299-308.\u003c/li\u003e\n\u003cli\u003eDideriksen, J. L., D. Farina, and R. M. Enoka. \u0026quot;Influence of Fatigue on the Simulated Relation between the Amplitude of the Surface Electromyogram and Muscle Force.\u0026quot; \u003cem\u003ePhilos Trans A Math Phys Eng Sci\u003c/em\u003e 368, no. 1920 (2010): 2765-81.\u003c/li\u003e\n\u003cli\u003eWhishaw, Ian Q., Valerie Berg dall, and Bryan Kolb. \u0026quot;Chapter 8 - Analysis of Behavior in Laboratory Rats11a Version of This Chapter Previously Appeared In: Whishaw, I.Q. Et Al. (1999). Analysis of Behavior in Laboratory Rodents. In Windhorst, U., Johansson, H. (Eds), Modern Techniques in Neuroscience Research, Pp. 1244\u0026ndash;1275, Heidelberg: Springer.\u0026quot; In \u003cem\u003eThe Laboratory Rat (Second Edition)\u003c/em\u003e, edited by Mark A. Suckow, Steven H. Weisbroth and Craig L. Franklin, 191-218. Burlington: Academic Press, 2006.\u003c/li\u003e\n\u003cli\u003eSporer, M. E., M. Aman, K. D. Bergmeister, D. Depisch, K. M. Scheuba, E. Unger, B. K. Podesser, and O. C. Aszmann. \u0026quot;Experimental Nerve Transfer Model in the Neonatal Rat.\u0026quot; \u003cem\u003eNeural Regen Res\u003c/em\u003e 17, no. 5 (2022): 1088-95.\u003c/li\u003e\n\u003cli\u003eLu, Wei, Jian-Ping Li, Zhen-Dong Jiang, Lin Yang, and Xue-Zheng Liu. \u0026quot;Effects of Targeted Muscle Reinnervation on Spinal Cord Motor Neurons in Rats Following Tibial Nerve Transection.\u0026quot; \u003cem\u003eNeural Regeneration Research\u003c/em\u003e 17, no. 8 (2022).\u003c/li\u003e\n\u003cli\u003eRaasveld, Floris V, Maximilian Mayrhofer-Schmid, Benjamin R Johnston, Barbara Gomez-Eslava, Yannick AJ Hoftiezer, Wen-Chih Liu, Ian L Valerio, and Kyle R Eberlin. \u0026quot;Targeted Muscle Reinnervation at the Time of Amputation to Prevent the Development of Neuropathic Pain.\u0026quot; \u003cem\u003eJournal of Plastic, Reconstructive \u0026amp; Aesthetic Surgery\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eBishay, Jeremy, Isobel Yeap, and Tim Wang. \u0026quot;The Effectiveness of Targeted Muscle Reinnervation in Reducing Pain and Improving Quality of Life for Patients Following Lower Limb Amputation.\u0026quot; \u003cem\u003eJournal of Plastic, Reconstructive \u0026amp; Aesthetic Surgery\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eBowen, J Byers, Corinne E Wee, Jaclyn Kalik, and Ian L Valerio. \u0026quot;Targeted Muscle Reinnervation to Improve Pain, Prosthetic Tolerance, and Bioprosthetic Outcomes in the Amputee.\u0026quot; \u003cem\u003eAdvances in Wound Care\u003c/em\u003e 6, no. 8 (2017): 261-67.\u003c/li\u003e\n\u003cli\u003eKuffler, Damien P., and Christian Foy. \u0026quot;Restoration of Neurological Function Following Peripheral Nerve Trauma.\u0026quot; \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e 21, no. 5 (2020): 1808.\u003c/li\u003e\n\u003cli\u003eWang, S., X. Liu, and Y. Wang. \u0026quot;Evaluation of Platelet-Rich Plasma Therapy for Peripheral Nerve Regeneration: A Critical Review of Literature.\u0026quot; \u003cem\u003eFront Bioeng Biotechnol\u003c/em\u003e 10 (2022): 808248.\u003c/li\u003e\n\u003cli\u003eAuger, F. A., L. Gibot, and D. Lacroix. \u0026quot;The Pivotal Role of Vascularization in Tissue Engineering.\u0026quot; \u003cem\u003eAnnu Rev Biomed Eng\u003c/em\u003e 15 (2013): 177-200.\u003c/li\u003e\n\u003cli\u003eGiglia, Giuseppe, Fernando Rosatti, Antonino Giulio Giannone, Giuditta Gambino, Maria Grazia Zizzo, Ada Maria Florena, Pierangelo Sardo, and Francesca Toia. \u0026quot;Vascularized Versus Free Nerve Grafts: An Experimental Study on Rats.\u0026quot; \u003cem\u003eJournal of Personalized Medicine\u003c/em\u003e 13, no. 12 (2023): 1682.\u003c/li\u003e\n\u003cli\u003eSaffari, T. M., M. Bedar, C. A. Hundepool, A. T. Bishop, and A. Y. Shin. \u0026quot;The Role of Vascularization in Nerve Regeneration of Nerve Graft.\u0026quot; \u003cem\u003eNeural Regen Res\u003c/em\u003e 15, no. 9 (2020): 1573-79.\u003c/li\u003e\n\u003cli\u003eXu, Y., Y. Yao, H. Lyu, S. Ng, Y. Xu, W. S. Poon, Y. Zheng, S. Zhang, and X. Hu. \u0026quot;Rehabilitation Effects of Fatigue-Controlled Treadmill Training after Stroke: A Rat Model Study.\u0026quot; \u003cem\u003eFront Bioeng Biotechnol\u003c/em\u003e 8 (2020): 590013.\u003c/li\u003e\n\u003cli\u003eSoendenbroe, C., M. F. Heisterberg, P. Schjerling, A. Karlsen, M. Kjaer, J. L. Andersen, and A. L. Mackey. \u0026quot;Molecular Indicators of Denervation in Aging Human Skeletal Muscle.\u0026quot; \u003cem\u003eMuscle Nerve\u003c/em\u003e 60, no. 4 (2019): 453-63.\u003c/li\u003e\n\u003cli\u003eDominguez, Elizabeth, Allisa Barber, Jose Lucas Zepeda, and Gwendolyn Hoben. \u0026quot;How Timing and Preexisting Pain Affect Outcomes of Tmr: A Systematic Review.\u0026quot; \u003cem\u003ePlastic and Aesthetic Research\u003c/em\u003e 11, no. 0 (2024): 53.\u003c/li\u003e\n\u003cli\u003eLauer, H., C. Prahm, J. T. Thiel, J. Kolbenschlag, A. Daigeler, D. Hercher, and J. C. Heinzel. \u0026quot;The Grasping Test Revisited: A Systematic Review of Functional Recovery in Rat Models of Median Nerve Injury.\u0026quot; \u003cem\u003eBiomedicines\u003c/em\u003e 10, no. 8 (2022).\u003c/li\u003e\n\u003cli\u003eVela, F. J., G. Mart\u0026iacute;nez-Chac\u0026oacute;n, A. Ballest\u0026iacute;n, J. L. Campos, F. M. S\u0026aacute;nchez-Margallo, and E. Abell\u0026aacute;n. \u0026quot;Animal Models Used to Study Direct Peripheral Nerve Repair: A Systematic Review.\u0026quot; \u003cem\u003eNeural Regen Res\u003c/em\u003e 15, no. 3 (2020): 491-502.\u003c/li\u003e\n\u003cli\u003eCarr, M. M., T. J. Best, S. E. Mackinnon, and P. J. Evans. \u0026quot;Strain Differences in Autotomy in Rats Undergoing Sciatic Nerve Transection or Repair.\u0026quot; \u003cem\u003eAnn Plast Surg\u003c/em\u003e 28, no. 6 (1992): 538-44.\u003c/li\u003e\n\u003cli\u003eFerreira, A., and M. Rapoport. \u0026quot;The Synapsins: Beyond the Regulation of Neurotransmitter Release.\u0026quot; \u003cem\u003eCell Mol Life Sci\u003c/em\u003e 59, no. 4 (2002): 589-95.\u003c/li\u003e\n\u003cli\u003eThiel, G. \u0026quot;Synapsin I, Synapsin Ii, and Synaptophysin: Marker Proteins of Synaptic Vesicles.\u0026quot; \u003cem\u003eBrain Pathol\u003c/em\u003e 3, no. 1 (1993): 87-95.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroengineering-and-rehabilitation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jner","sideBox":"Learn more about [Journal of NeuroEngineering and Rehabilitation](http://jneuroengrehab.biomedcentral.com/)","snPcode":"12984","submissionUrl":"https://submission.nature.com/new-submission/12984/3","title":"Journal of NeuroEngineering and Rehabilitation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Targeted Nerve Function Replacement (TNFR), Electromyography (EMG), Autophagic Behavior, Spinal Cord, Dorsal Root Ganglion (DRG)","lastPublishedDoi":"10.21203/rs.3.rs-5460332/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5460332/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTargeted Muscle Reinnervation (TMR) improves real-time control of EMG-based prostheses by connecting severed nerves to adjacent muscles, creating new EMG signals. However, TMR requires cutting original nerve connections, which can cause denervation atrophy and limit functional recovery. As an alternative, Targeted Nerve Function Replacement (TNFR) offers promising potential for limb function restoration. This study evaluates TNFR efficacy in restoring denervated muscle function across different postoperative intervals in a rat model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThirty Sprague-Dawley rats (220\u0026ndash;250 g) were divided into five equal groups (n\u0026thinsp;=\u0026thinsp;6 per group): control (no transection), denervation (transection without repair), immediate TNFR after median nerve transection, 2-week delayed TNFR, and 4-week delayed TNFR. The median nerve was selected for reinnervation with the musculocutaneous nerve innervating the brachialis muscle serving as the anastomosis target. All assessments were conducted 4 weeks post-TNFR intervention, including intramuscular bipolar EMG recordings (1024 Hz sampling rate), behavioral assessment, muscle tension measurement, dorsal root ganglia (DRG) histology, and spinal cord motor neuron evaluation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings revealed that immediate TNFR significantly outperformed delayed interventions across all measured parameters. EMG amplitude and root mean square values were significantly higher in the immediate TNFR group compared to delayed intervention groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, maximum contraction force and maximum tetanic contraction force of the biceps brachii demonstrated significantly superior recovery in the immediate TNFR group versus delayed groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Histological examination confirmed significantly greater preservation of sensory neurons in the DRG following immediate TNFR compared to delayed interventions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Immunofluorescence analysis showed that immediate TNFR better preserved synaptic protein expression (synaptophysin/SYN) in motor neurons of the spinal cord compared to delayed interventions, indicating enhanced preservation of motor neuron function. Notably, immediate TNFR prevented the autophagic behavior observed in rats with delayed or absent intervention, suggesting better neuropathic pain prevention.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTiming critically influences TNFR outcomes, with immediate intervention yielding optimal restoration of both motor and sensory functions. This study provides valuable insights for optimizing surgical strategies in peripheral nerve injury, with important implications for limb reconstruction, rehabilitation protocols, and prosthetic development.\u003c/p\u003e","manuscriptTitle":"Improving Nerve and Muscle Function: An Exploration of Targeted Nerve Function Replacement Following Differential Delay Periods in a Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 04:31:25","doi":"10.21203/rs.3.rs-5460332/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-18T02:35:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-30T18:34:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-19T19:06:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311862839677336965131387475357476057899","date":"2025-04-03T13:02:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276294849809423686997900210861303361582","date":"2025-04-03T01:06:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T00:45:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T23:09:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of NeuroEngineering and Rehabilitation","date":"2025-03-30T07:46:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroengineering-and-rehabilitation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jner","sideBox":"Learn more about [Journal of NeuroEngineering and Rehabilitation](http://jneuroengrehab.biomedcentral.com/)","snPcode":"12984","submissionUrl":"https://submission.nature.com/new-submission/12984/3","title":"Journal of NeuroEngineering and Rehabilitation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b51e88b-cc70-48fd-93ac-e03317697c2d","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:03:18+00:00","versionOfRecord":{"articleIdentity":"rs-5460332","link":"https://doi.org/10.1186/s12984-025-01666-0","journal":{"identity":"journal-of-neuroengineering-and-rehabilitation","isVorOnly":false,"title":"Journal of NeuroEngineering and Rehabilitation"},"publishedOn":"2025-07-04 15:57:06","publishedOnDateReadable":"July 4th, 2025"},"versionCreatedAt":"2025-04-03 04:31:25","video":"","vorDoi":"10.1186/s12984-025-01666-0","vorDoiUrl":"https://doi.org/10.1186/s12984-025-01666-0","workflowStages":[]},"version":"v1","identity":"rs-5460332","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5460332","identity":"rs-5460332","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}