Fu's under-skin needling may help muscle repair by balancing energy production in rats with long-term nerve pain

Twenty-four adult Sprague–Dawley rats (200–250 g, 8–10 weeks old) were acclimatized and isolated, housed in ventilated rooms with controlled humidity and temperature (22°C–24°C), under a normal 12-h light/dark cycle, with free access to food and water. All experiments were approved by the Animal Care and Use Committee of the Chang Bing Show Chwan Memorial Hospital (IACUC approved number: #111031).The rats were randomly divided into the sham, CCI, and CCI + FSN groups (n = 8 for each group).

The CCI model was established by modifying the method developed by Bennett et al., in 1988 for chronic constriction injury (CCI) of the sciatic nerve. The rats were anesthetized under 4% isoflurane induction, followed by maintenance with 1%–2% isoflurane. The right sciatic nerve was carefully exposed and tied using 3–0 chromic catgut sutures. The distance between the sutures was approximately 1 mm, and the tightness of all the ligatures was the same. Finally, after ligation, the nerve was returned to its original position, and the muscle and skin layers were sutured. The sham operations involved the exposure of the sciatic nerve without ligation. Pain thresholds were measured on days 1, 3, 5, and 7 after modelling. The presence of neuropathic pain indicated successful modeling, and rats that did not develop neuropathic pain were excluded. The intervention began on the first day after successful modeling (day 8 of the total timeline). The FSN group received FSN needle treatment at fixed time points on days 8, 10, 12, and 14. The treatment frequency and timing of FSN administration were determined based on both our previous clinical and preclinical studies (Chiu et al., 2023a; Chiu et al., 2022; Chiu et al., 2023b; Chiu et al., 2024). On day 14, all animals were euthanized under deep anesthesia. Skeletal muscles from the ipsilateral side—including the vastus lateralis, biceps femoris, and gastrocnemius—were carefully dissected for further analysis. These specific muscles were selected based on their anatomical relationship with the sciatic nerve and were used in subsequent gross morphology assessment, muscle weight quantification, and histological or molecular evaluations.

The 8–10-week-old Sprague–Dawley rats were acclimatized, quarantined, and kept in ventilated humidity- and temperature-controlled rooms with a 12-h light/dark cycle. All experiments were approved by the Animal Care and Use Committee of the Chang Bing Show Chwan Memorial Hospital (IACUC approved number: #111031). Twenty-four animals were randomly assigned to three groups: sham, CCI and CCI + FSN. The CCI model was established in a manner similar to the method described (Chiu et al., 2024). Briefly, the right sciatic nerve was loosely ligated using a 3–0 chromic gut. A sham surgery was performed by exposing the sciatic nerve without ligation. All surgical procedures were performed under anesthesia with 4% isoflurane for induction and 1%–2% isoflurane for maintenance.

Disposable needles were used in the FSN group (Nanjing Paifu Medical Science and Technology Co., Nanjing, China). Without anesthesia, the rat hind limb was extended to tension and the FSN needle was inserted towards the tightened muscle (muscle with Myofascial Trigger Points, approximately near the biceps femoris muscle). The needle was rapidly advanced to prevent tension. After the needle entered the skin, the swaying movement began. This movement is a smooth, gentle, and fan-shaped swaying using the thumb as the pivot. The index, middle, and ring fingers were aligned in straight lines. The middle and thumb faced each other during insertion, whereas the index and ring fingers alternated back and forth. The frequency of this movement was 100 times per minute, with each session lasting approximately 1 min. The procedure was performed every 2 days for a total of four sessions (on days 8, 10, 12, and 14).

The rats were placed in a box on a metal mesh, with eight rats per group. They were left to acclimate for 15–30 min until they remained calm in an unfamiliar environment. Each rat was then placed individually in a small cage with a framework and allowed to acclimatize for 5 min before testing began. Von Frey filaments were applied to the ipsilateral hindpaw until it bent for 5 s. The range of the filaments used was 0.008–300 g. A positive response was considered if the animal withdrew or shook its hindpaw in at least four of five applications. The first step in the Von Frey method is to estimate the response of a filament close to the 50% withdrawal threshold (Chiu et al., 2023b). If no response was observed, a filament with a greater force was used; conversely, if the response was positive, a filament with a lower force was used. This process was continued until four readings following the first crossover response were recorded. Although the responses were near the threshold, the calculation detected a 50% threshold.

After euthanasia on day 14, the ipsilateral vastus lateralis, biceps femoris, and gastrocnemius muscles were carefully excised, fixed in 10% neutral-buffered formalin for 48 h, and then dehydrated and embedded in paraffin. Longitudinal sections (as shown in Figures 3A–C) with a thickness of 5 μm were cut using a microtome and mounted on glass slides. Sections were stained with hematoxylin and eosin (H&E) according to standard protocols. Briefly, slides were deparaffinized, rehydrated, stained with hematoxylin for nuclear visualization, followed by eosin for cytoplasmic contrast, then dehydrated and mounted.

For cross-sectional area (CSA) analysis, digitized H&E-stained images were captured using a light microscope (Leica) at ×200 magnification. CSA was quantified using ImageJ software. At least eight randomly selected non-overlapping fields per muscle section were analyzed per animal, and individual muscle fiber cross-sectional areas (in arbitrary units, a.u.) were measured manually by outlining fibers using the polygon selection tool. A minimum of 100 fibers per muscle per animal were included in the final analysis.

TEM was used to visualize cellular structures. A JEOL JEM-1400 electron microscope operating at 120 keV and equipped with an Ultrascan CCD Camera System (Gatan) was used. To ensure unbiased morphometric analysis of organelles, rat cells were experimentally manipulated and fixed by a single researcher. Another researcher who was blinded to the experimental conditions processed and collected images randomly using an electron microscope. Quantitative analysis was performed on anonymized samples by a third researcher who received random images of whole cells at all magnifications.

Mitochondria were counted using the ImageJ software (version 1.54, National Institutes of Health, United States). Healthy mitochondria were characterized by clear, uniform cristae, and intact, distinct outer and inner membranes. Damaged mitochondria were identified as those with uneven or absent cristae, ruptured outer membranes, or significant swelling. Mitochondrial content was measured from images at ×10,000 magnification and expressed as the mitochondrial count per field. For the statistical analysis, data from 20 microscopic photographs of the control, CCI, and CCI + FSN groups were included.

Glycogen storage was quantified manually in each photograph and defined as the number of glycogen granules observed in the TEM field of view. Researchers blinded to the group assignments captured TEM images at ×10,000 magnification across eight different fields of view and manually counted the glycogen granules. The density of glycogen granules (granules/μm²) was measured and statistically compared between groups.

For RNA extraction of the sciatic nerves from rats, tissues were frozen at–80°C, and total RNA was extracted using 1 mL of REzolTM C&T reagent (PROtech Technologies, Inc., Taipei, Taiwan, R.O.C.). The phenol–chloroform phase was used for protein isolation according to the manufacturer's instructions. RNA was extracted using an RNeasy Plus Universal Kit (Qiagen). Library preparation, RNA sequencing, and bioinformatic analyses were performed at the Functional Genomics Center, Zurich. Libraries were prepared using the TruSeq RNA Stranded Library Prep Kit (Illumina, Inc., San Diego, CA, United States), and sequencing was performed usingan Illumina NovaSeq 6000 instrument with 150 bp paired-end reads.

For all samples, the read quality was assessed using FastQC. The data generated from the sequencer were passed through the bcl2fastq conversion program to convert the binary base calls into a common FASTQ format. This FASTQ file is referred to as the raw data. Subsequently, low-quality bases, adapters, and reads containing many unknown N bases were removed from the raw data using FASTP to obtain clean reads. To remove RNA, the reads that filtered out rRNA from the reference genome were compared. Genes with a false discovery rate of <0.05 were defined as differentially expressed, and both log2 fold change and p < 0.01 were used to perform a series of analyses, such as gene ontology (GO), gene set enrichment analysis (GSEA), and function enrichment.

Western blotting was performed as previously described (Liu et al., 2022). Briefly, total protein was extracted from cells using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined by BCA assay, and equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate HRP-conjugated secondary antibodies. The bands were visualized using enhanced chemiluminescence (ECL). Details of the antibodies used are provided in Table 1.

The ADP/ATP ratio was calculated using the ApoSENSOR™ ADP/ATP ratio bioluminescence assay kit (cat. no. K255-200), and anadenosine assay kit (cat. no. K327) was purchased from BioVision, Inc. Fresh muscle tissue samples from the vastus lateralis, biceps femoris, and gastrocnemius were collected, and single-cell suspensions were prepared using a 70-μm sieve to confirm cell counts. Briefly, the ADP/ATP ratio assay required 1 × 10^6 cells to be added to a 96-well luminescence plate along with a nucleotide-releasing buffer, following the manufacturer's instructions. The ADP/ATP ratio was calculated using the formula: ADP/ATP ratio = (C−B)/A.As described above, 10 μL of undiluted muscle sample was mixed with the adenosine assay buffer for adenosine detection, following the manufacturer's instructions. The sample adenosine concentration (C) was calculated using the formula: C = B/V × D pmol/μ, and the result was normalized to protein concentration to obtain the final measurement. An AMP assay kit (ab273275) was purchased from Abcam. Muscle tissue samples were processed at a protein concentration of 10 mg, as specified by the manufacturer.

Skeletal muscle tissue was quickly cryopreserved after isolation, and enzyme-linked immunosorbent assay (ELISA) was performed using a commercially available ELISA kitswith FGF (MFB00, R&D Systems), VEGF (RAB0512, Sigma), NGF (MBS2709258, MyBioSource), and IGF (MG100, R&D Systems). According to the manufacturer's instructions, the optical density was measured at 450 nm usinga plate reader, and the sample values were calculated from the standard curve.

Publicly available gene expression data were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/↗) under the accession number GSE9103. This dataset contains transcriptomic profiles of vastus lateralis muscle biopsies obtained from young and elderly individuals, categorized into sedentary and endurance-trained groups. The following genes of interest were analyzed based on their relevance to mitochondrial autophagy and muscle regeneration: PARK2, BNIP3L, VDAC1, VDAC2, FGFR1, FGFR3. Comparisons were made among the four groups: sedentary young, trained young, sedentary old, and trained old.

Statistical analyses were performed as previously described (Cheng et al., 2023). Briefly, all data are presented as means ± standard deviations (SD). Statistical comparisons among three groups were conducted using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test to determine intergroup differences. A p-value of less than 0.05 was considered statistically significant. GraphPad Prism version (version 7.0) was used for all statistical analyses and graph generation.

To investigate the effects of FSN on muscle mass and mechanical withdrawal thresholds in the CCI rat model, we first established a timeline for the experimental procedures (Figure 1A). After acclimation, the rats underwent the CCI procedure on day 0, with FSN treatment initiated on day 7 and continued until day 14. Mechanical, histological, and molecular analyses were performed during the final assessment phase (days 11–14). The mechanical withdrawal threshold, a measure of neuropathic pain, was significantly lower in the CCI group than in the sham group, indicating successful establishment of the CCI model. FSN treatment partially restored the withdrawal threshold (Figure 1B). The anatomical diagram (Figure 1C) illustrates the relationship between the sciatic nerve and its associated muscles. Representative images of the vastus lateralis, biceps femoris, and gastrocnemius muscles showed marked atrophy in the CCI group, which was alleviated by FSN treatment (Figure 1D). Quantitative analysis revealed a significant reduction in the relative weights of the vastus lateralis and gastrocnemius muscles in the CCI group compared to those in the sham group, which was reversed by FSN treatment (Figure 1E). These findings demonstrated the protective effects of FSN against CCI-induced muscle atrophy and neuropathic pain.

Histological analysis using hematoxylin and eosin staining revealed distinct morphological changes in muscle fibers across groups (Figures 2A–C). The vastus lateralis and gastrocnemius muscles in the CCI group displayed significant atrophy and disorganization of muscle fibers compared to those in the sham group. Treatment with FSN ameliorates these structural abnormalities. Quantitative CSA analysis supported these findings, showing significant reductions in CSA in the vastus lateralis and gastrocnemius muscles in the CCI group compared to the sham group, while biceps femoris no significantly change (Figure 2D). FSN treatment restored the CSA to levels comparable to those in the sham group. Thus, FSN treatment may preservethe structure and integrity of CCI-affected muscles.

Evaluation of mitochondrial status in the vastus lateralis based on the action site of FSN using transmission electron microscopy (TEM). TEM reveals ultrastructural changes in the longitudinal section (Figure 3A) and transverse section (Figure 3B) of vastus lateralis muscle fibers. At lower magnifications, the CCI group exhibited mitochondrial damage including swelling and vacuolation, which was absent in the sham group. Higher magnification highlighted the accumulation of autophagosomes and disrupted glycogen storage in the CCI group (Figure 3C). Treatment with FSN mitigated these changes, restored mitochondrial integrity, and reduced autophagosome formation. The quantitative analysis further supported these observations. The number of autophagosomes per field was significantly increased in the CCI group compared to that in the sham group and was reduced by FSN treatment (Figure 3D). Although mitochondrial density was significantly lower in the CCI group than in the sham group, this recovered with FSN treatment (Figure 3E). Glycogen particle density also decreased in the CCI group and improved with FSN treatment (Figure 3F). Thus, FSN may protect against CCI-induced mitochondrial dysfunction and promote glycogen preservation.

RNA sequencing analysis was performed on three muscle groups on the nerve-ligated side 2 weeks after the completion of the CCI ligation surgery. Differential gene expression analysis identified significant transcriptional changes in the biceps femoris, vastus lateralis, and gastrocnemius muscles under CCI conditions compared to those in the sham group (Figure 4A), and volcano plot as showed in the Supplementary Figures S1A-C. Pathway enrichment analysis highlighted pathways related to neurodegeneration, oxidative phosphorylation, and mitophagy in the vastus lateralis muscle (Figure 4B). These results suggest altered mitochondrial function and energy metabolism in response to CCI. Similar pathways including PI3K–Akt, MAPK, and AMPK signalingwere enriched in the biceps femoris and gastrocnemius muscles (Figures 4C,D). These pathways reflect the key biological processes affected by CCI including energy metabolism, stress response, and muscle repair. These findings underscore the systemic effect of CCI on muscle tissues and the potential for targeted interventions to mitigate these effects.

To elucidate the potential molecular mechanisms of FSN treatment for CCI, further RNA sequencing analysis was conducted on the three muscle groups in the CCI + FSN and CCI groups. However, we found no significant differences in the biceps femoris between the CCI + FSN and CCI groups. In contrast, significant differences were observed in the vastus lateralis and gastrocnemius muscles, and principal component analysis as showed in the Supplementary Figures S2A. Significant differences genes as showed in the Supplementary Figures S1B,C. For the vastus lateralis, functional molecular signals by CCI + FSN vs CCI resulted in significant changes in various biological processes, molecular functions, and cellular components, as identified through GO enrichment analysis (Figure 5A). Key enriched biological processes included mitochondrial organization and inflammatory response, whereas molecular functions, such as actin cytoskeleton binding and ATPase activity were significantly modulated. Cellular component analysis highlighted mitochondrial-related structures and extracellular matrix components. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Figure 5B) revealed the significant involvement of pathways such as oxidative phosphorylation, MAPK signaling, and lipid metabolism. The gene concept network (Figure 5C) demonstrated key associations between the enriched biological processes and genes, identifying functional modules prominently influenced by FSN. Additionally, the protein–protein interaction (PPI) network (Figure 5D) emphasized the interconnectivity and functional relationships among the enriched genes. GSEA further supported these findings, showcasing notable biological processes modulated by FSN (Figures 5E–J). Specifically, mitochondrial nucleoid organization (NES = 2.30, p = 2.48E−06) and protein localization in the mitochondria (NES = 1.85, p = 9.5E−06) were upregulated, indicating enhanced mitochondrial activity. Similarly, ATP synthesis-coupled electron transport (NES = 1.36, p = 2.18E−03) and myosin filament organization (NES = 1.85, p = 1.54E−03) were significantly enriched. Conversely, inflammatory response (NES = −2.12, p = 2.56E−09) was downregulated, suggesting an anti-inflammatory effect of FSN.

In the gastrocnemius muscle, FSN also led to notable changes in biological processes, molecular functions, and cellular components (Figure 6A). Biological processes, such as muscle organ development and extracellular matrix organization, were enriched. Molecular functions including the structural constituents of muscle and actin binding were modulated, whereas cellular component analysis highlighted collagen-containing extracellular matrix and contractile fibers. KEGG pathway analysis (Figure 6B) revealed significant pathways including oxidative phosphorylation, extracellular matrix organization, and cell adhesion. The geneconcept network (Figure 6C) and PPI network (Figure 6D) further highlighted the key functional modules and interconnectivity among the enriched genes. The GSEA results (Figures 6E–J) demonstrated that FSN enhanced biological processes such as actin filament bundle organization (NES = 1.96, p = 9.35E−05), muscle cell development (NES = 1.50, p = 3.27E−03), and regeneration (NES = 1.43, p = 3.55E−03). Additionally, positive regulation of glycogen biosynthetic processes (NES = 1.94, p = 2.63E−04) was observed, indicating improved energy metabolism. Similar to the vastus lateralis, FSN downregulated inflammatory pathways, as evidenced by the regulation of interleukin-1 beta production (NES = −1.73, p = 2.45E−04). These results highlight the significant role of FSN in modulating key biological processes and pathways in both the vastus lateralis and gastrocnemius muscles. FSN enhances mitochondrial function, energy metabolism, and muscle regeneration, while mitigating inflammatory responses, underscoring its therapeutic potential for managing CCI.

Finally, we evaluated changes in energy metabolism and protein expression across the experimental groups. Quantification of the ADP/ATP ratio, AMP levels, and adenosine levels revealed significant disruptions in energy homeostasis in the CCI group compared to that in the sham group, which was ameliorated by FSN treatment (Figures 7A–C). Meanwhile, in the vastus lateralis and gastrocnemius muscles, FSN treatment significantly increase FGF, NGF and IGF compared to that in the CCI group, but VEGF not significant, showed in the Supplementary Figures S3A-D. Western blot analysis of key proteins involved in energy metabolism and stress responses showed altered expression of FGFR1, FGFR3, HSP60, PINK1, VDAC, BNIP3L, phosphorylation-ERK, and phosphorylation-FOXO3 in the CCI group (Figure 7D). Among them, the most significant changes were observed in the vastus lateralis and gastrocnemius muscles. Compared to the sham group, the CCI group exhibited increased expression of autophagy-related markers HSP60, PINK1, VDAC, and BNIP3L, while showing reduced activation of growth factor-related pathways, including FGFR1, FGFR3, phosphorylation-ERK, and phosphorylation-FOXO3. However, compared to the CCI group, the CCI + FSN group demonstrated a significant reduced BNIP3L expression and an increase FGFR1, FGFR3, phosphorylation-ERK, and phosphorylation-FOXO3, without affecting the expression of HSP60, PINK1, or VDAC. Therefore, FSN treatment restored the expression levels of these proteins toward those observed in the sham group, indicating their role in maintaining mitochondrial function and the cellular stress response. Collectively, these findings demonstrated that FSN treatment effectively mitigated CCI-induced disruptions in muscle energy metabolism and protein expression, highlighting its therapeutic potential in neuropathic conditions.

Prolonged sedentary lifestyles are a significant factor contributing to nerve compression. To assess the impact of nerve compression on the vastus lateralis muscle, a further analysis of the research database focusing on age and sedentary behavior grouping was conducted. The data for this analysis were obtained from the publicly available GSE9103 dataset. In the mitophagy-related genes, A significant decrease in PARK2 expression was observed in trained old individuals compared to their sedentary counterparts (p < 0.05), no significant differences were observed between the young groups (Figure 8A). The expression of BNIP3L and VDAC1 were significantly upregulated in sedentary older individuals compared to trained young individuals (p < 0.05) (Figures 8B,C). No significant differences were detected in VDAC2 expression across all groups (Figure 8D). Among the growth-related genes, FGFR1 levels in the sedentary elderly group showed a slight decrease compared to the trained young group (73.24 ± 3.464 vs 80.65 ± 4.610; p = 0.215) (Figure 8E). Similarly, FGFR3 demonstrated a comparable trend, with lower levels observed in the sedentary elderly group compared to the trained older group (27.74 ± 2.324 vs 32.93 ± 3.578; p = 0.240). These results suggest that a sedentary lifestyle, one of the contributing factors to sciatica, may influence the expression of specific mitophagy-related genes, such as PARK2 and BNIP3L, which exhibit differential expression in response to sedentarism and aging. In contrast, growth-related genes, such as FGFR1 and FGFR3, are mildly decrease. This finding may have important implications for developing interventions to optimize skeletal muscle health in the context of sciatica.