Skip to main content
NCBI home page
Lippincott Open Access logoLink to Lippincott Open Access
. 2024 Oct 24;110(12):7563–7572. doi: 10.1097/JS9.0000000000002128

Low-energy extracorporeal shockwave therapy improves locomotor functions, tissue regeneration, and modulating the inflammation induced FGF1 and FGF2 signaling to protect damaged tissue in spinal cord injury of rat model: an experimental animal study

Chieh-Cheng Hsu a,b,c, Kay LH Wu d, Jei-Ming Peng d, Yi-No Wu e, Hou-Tsung Chen a,b, Meng-Shiou Lee f, Jai-Hong Cheng a,g,*
PMCID: PMC11634128  PMID: 39453843

Abstract

Background:

Spinal cord injury (SCI) is a debilitating condition that results in severe motor function impairments. Current therapeutic options remain limited, underscoring the need for novel treatments. Extracorporeal shockwave therapy (ESWT) has emerged as a promising noninvasive approach for treating musculoskeletal disorders and nerve regeneration.

Methods:

This study explored the effects of low-energy ESWT on locomotor function, tissue regeneration, inflammation, and mitochondrial function in a rat SCI model. Experiments were performed using locomotor function assays, CatWalk gait analysis, histopathological examination, immunohistochemical, and immunofluorescence staining.

Results:

The findings demonstrated that low-energy ESWT had a dose-dependent effect, with three treatment sessions (ESWT3) showing superior outcomes compared to a single session. ESWT3 significantly improved motor functions [run patterns, run average speed, and maximum variation, as well as the Basso, Beattie, and Bresnahan score] and promoted tissue regeneration while reducing inflammation. ESWT3 significantly decreased levels of IL-1β, IL6, and macrophages (CD68) while increasing leukocyte (CD45) infiltration. Additionally, ESWT3 upregulated NueN and mitofusin 2 (MFN2), suggesting enhanced neuronal health and mitochondrial function. Moreover, ESWT3 modulated the expression of fibroblast growth factor 1 (FGF1), FGF2, their receptor FGFR1 and phosphorylation of ERK, aiding tissue repair, and regeneration in SCI.

Conclusions:

This study highlights the potential of low-energy ESWT as an effective noninvasive treatment for SCI, demonstrating significant improvements in motor recovery, tissue regeneration, anti-inflammatory effects, and mitochondrial protection. These findings provide valuable insights into the mechanisms of ESWT and its therapeutic application for SCI recovery.

Keywords: extracorporeal shockwave therapy, FGF signaling, locomotor function, spinal cord injury, tissue regeneration

Introduction

Highlights

  • Extracorporeal shockwave therapy (ESWT) with three treatments is better than once for spinal cord injury.

  • ESWT promotes locomotor function in rats with spinal cord injury.

  • ESWT increases the expression of NueN and MFN2 in the cells of spinal cord injury.

  • ESWT reduces inflammation induced FGF1 and FGF2.

Spinal cord injury (SCI) occurs due to traumatic injury to the spinal cord, resulting in complete or incomplete impairment of neural functions, affecting mobility and sensory function1. The damages caused by SCI is categorized into primary and secondary injury2. The primary injury refers to the immediate mechanical trauma to spinal cord. It typically involves bruising of the spinal cord, followed by compression caused by dislocated bone and soft tissues3. As the injury develops, the spinal cord may become rotated, lacerated, hyper-bent, and over-stretched; however, the white matter is usually spared2,3. The secondary damage occurs after the initial trauma, driven by the inflammatory response that regulates numerous cellular and molecular interactions4. This response activates and recruits peripheral and resident inflammatory cells, including monocytes, astrocytes, microglial cells, neutrophils, and T lymphocytes to further promoting the development of secondary damage following SCI5. The ultimate changes after SCI, including neuronal apoptosis, syrinx formation, and glial scar formation, can result in permanent loss of sensorimotor functions, often unresponsive to therapy6. Effective SCI treatment requires stabilizing the spine and managing inflammation to prevent further damage. Given the complexity of secondary injury cascades, a detailed understanding of these processes is crucial for selecting the optimal time-point for therapeutic interventions7. To date, researchers have explored various approaches to SCI treatment, including stem cell transplantation, bioengineering methods, exoskeletons, brain-computer interfaces, and functional electrical stimulation8,9. These advanced methods offer promising prospects for the future treatment of SCI.

One emerging noninvasive treatment option that has gained attention for its therapeutic potential in SCI is extracorporeal shockwave therapy (ESWT). ESWT has shown effects in treating orthopedic disorders, including tendinopathies and nonunion of long-bone fractures, as well as soft tissue for over 30 years10. Many studies have demonstrated that ESWT stimulated sub-periosteal callus formation by inducing micro-fraction in the cortex11. Unlike many existing treatment methods that require invasive procedures or carry risks of severe side effects, ESWT offers a less intrusive approach. Additionally, ESWT has been reported to promote the expression of growth factors, including bone morphogenetic protein and vascular endothelial growth factor (VEGF)12–14. It also improves blood supply, stimulates cell proliferation, and promotes tissue regeneration positions it as a promising solution for addressing the multifaceted pathophysiology of SCI13,15,16. According to these characteristics, ESWT is potential for reducing inflammation and promoting nerve regeneration makes it a valuable addition to the range of therapeutic options for SCI.

Studies have shown that ESWT (2000 impulses, 0.18 mJ/mm²) can stimulate new bone growth and significantly increased the stiffness of spinal fusion segments in flexion and extension in animal model. These results support the efficacy and safety of ESWT in promoting spinal fusion in animals17–19. Additionally, although ESWT has been shown to induce microscopic alterations in myelin sheaths in treated spinal cords, it does not result in neurological symptoms20,21. Furthermore, studies on the effects of ESWT on the sciatic nerve in rats have demonstrated that high-energy shockwaves (2000 impulses, 0.49 mJ/mm²) can lead to demyelination and a decrease in motor nerve conduction velocity. In contrast, low-energy shockwave treatments (2000 impulses, 0.08 mJ/mm²) showed no significant differences in nerve structure or function, indicating a safer profile22. Additionally, ESWT has been reported to promote recovery in cases of sciatic nerve crush injury by enhancing peripheral nerve regeneration23,24. Importantly, all observed changes following low-energy ESWT were reversible, suggesting a low risk of long-lasting or harmful complications in peripheral nerves, further supporting its safety for therapeutic use22.

The effects of ESWT on different cell types and tissues can influence both intact or damaged nerve cells and tissue. Most studies have focused on the palliative effects of low-energy ESWT in both clinical and experimental applications25–27. When applied to the skin at effective doses, ESWT has been shown to induce analgesia; however, it may also result in localized nerve injury, which stimulates the expression of regeneration and transcription-related factors in dorsal root ganglion neurons28. Several studies have indicated that an appropriate dose of ESWT can promote cell proliferation, differentiation, and tissue regeneration. For example, a single treatment of 300 impulses at 0.1 mJ/mm² did not induce axonal degeneration after 1 week, whereas treatments with 900 or 1500 impulses caused moderate to severe degeneration23. Recently, Yamaya et al.29 demonstrated that low-energy ESWT is a safe and effective therapeutic strategy for SCI. ESWT was found to enhance the expression of VEGF and Flt-1 in the spinal cord, reduce neuronal loss in injured neural tissue, and improve locomotor function following SCI29. However, further research is needed to determine the optimal dosage and to clarify the mechanisms of action of ESWT in the treatment of SCI. ESWT has been demonstrated as a noninvasive treatment method across various fields. Given the structural similarities between the spinal cord and the peripheral nervous system in both rats and humans, low-energy ESWT may provide similar beneficial effects in SCI treatments. In this study, a rat clip compression model was used to induce SCI, and the efficacy of low-energy ESWT was investigated with one or three treatments. The study also explored the motor function, pathological changes associated with SCI in neuron and mitochondria, as well as assessed the effects of low-energy ESWT on inflammation-induced fibroblast growth factor 1 (FGF1), FGF2, FGFR1, and phosphorylation of ERK (pERK) levels to protect the damaged spinal cord.

Materials and methods

Animals

A total of 40 with body weights ranging from 250 to 300 g of Sprague-Dawley (SD) rats (BioLASCO) were used in the experiments. The detailed experimental protocol of the animal study was approved by the Animal Care Committee of the hospital. The animals were maintained at the Laboratory Animal Center for 1 week before experiments. The rats were housed at 23±1°C with a 12 h light and dark cycle and given food and water. The work was reported in accordance with the Animals in Research: Reporting In Vivo Experiments guidelines30.

Spinal cord injury (SCI)

The 250–300 g of SD rats were anesthetized with Zoletil (25 mg/kg), along with Xylazine (10 mg/kg). Before surgery, the animals received a subcutaneous injection of 3 ml of saline. They were then placed on a heating pad set to 37°C during the surgical procedure, which involved performing a laminectomy at the T9 level to expose the spinal cord. Dissection of the extradural plane between the dura and adjacent vertebrae was carried out using a dissecting hook with a curvature and thickness similar to that of the clip. The SCI was induced using an applicator to apply a twice (once per minute) pressure of 60 g via a clip, which was then rapidly released to create acute impact compression injury31. The clip was allowed to compress the spinal cord for 1 min before being removed using the applicator. Postsurgery, ampicillin (25 mg/kg) and ketorolac (1 mg/kg/day) were administered for 5 days to prevent infection and alleviate pain.

Study design

Thirty two rats were randomized into four groups (eight rats for each group), as shown in Figure 1A. Sham group was that rats did not undergo surgery or receive treatment. The purpose of the Sham group is to differentiate between the effects of surgery-related stress and the actual injury, ensuring that any observed changes in the experimental groups are specifically due to the SCI and not external procedural factors. SCI group was that rats received clip compression on the T9 spinal cord to create injury. This group allows us to observe the natural progression of SCI and serves as a comparison to determine the effectiveness of the treatments in the other groups. It provides a baseline to assess the impact of ESWT on SCI recovery. In the ESWT group, the SCI rats were treated with shockwaves (0.13 mJ/mm2 with 500 impulses, 4 Hz), which were modified and from a shockwave treatment apparatus (DUOLITH SD1, STORZ MEDICAL AG, Swiss) post-one week surgery32. The purpose is to investigate whether a single treatment can reduce inflammation, promote nerve regeneration, and improve functional recovery following SCI. This group helps assess the short-term effects of ESWT on SCI. In the ESWT3 group, the SCI rats received shockwaves at 1, 2, and 3 weeks postsurgery. The objective is to determine whether multiple sessions of ESWT lead to enhanced therapeutic effects compared to a single treatment. By comparing this group with the ESWT group, we aim to explore whether repeated applications of ESWT provide additional benefits in terms of reducing inflammation, improving tissue repair, and enhancing recovery outcomes. All rats were sacrificed at 7 weeks and the specimens were collected for further analysis.

Figure 1.

Figure 1

Open in a new tab

The study design and locomotor function assay. (A) The graph presents the study design of the experiment including Sham, SCI, ESWT, and ESWT3 groups. ESWT is that shockwaves applied at 1 week postsurgery and ESWT3 is that shockwaves applied at 1, 2, and 3 weeks postsurgery, respectively. The sacrifice of animals are at 6 weeks postsurgery. Eight rats are used for each group. (B) The injury is created at T9 of the spinal cord in the rat as indicated by the black arrow. (C) The damage region of the spinal cord is indicated by the black arrow. (D) The hind legs of the rat drag in the SCI and ESWT groups. Some of the rats have their paws turned upwards in the ESWT3 group at 6 weeks postsurgery. (E) The BBB scores are presented in the Sham, SCI, ESWT, and ESWT3 groups at 2, 3, 5, and 7 weeks. * P<0.05 compared between ESWT and SCI groups. # P<0.05 and ## P<0.01 are compared between ESWT3 and SCI group. § P<0.05 compared between ESWT and ESWT3 group. BBB, Basso, Beattie, and Bresnahan; ESWT, extracorporeal shockwave therapy; ESWT3, three ESWT treatments; N=8; SCI, spinal cord injury.

Locomotor function assay

The limb motor functions were assessed at 2, 3, 5, and 7 weeks using the open-field locomotor test developed by Basso, Beattie, and Bresnahan (BBB)33. The score of each hind limb was recorded and the averages were calculated.

CatWalk gait analysis

The walking patterns of the rats were acquired and analyzed to measure the conditioned locomotion using the CatWalk Device (Noldus Information Technology Inc.). The four paws of rats were captured in the location of the locomotion by CatWalk Device. After an acclimatization period, baseline performance was assessed for the animals in the Sham, SCI, SW, and SW3 groups. A measurable run was defined as the ability of the animals to take three or more consecutive steps without interruption while traversing the walkway, and average values were subsequently calculated for analysis. The label paw prints of rats were adjusting the intensity threshold and autoclassifying the prints by CatWalk software (ver. 10.1). All steps of rats were detected between these two points.

Histopathological examination

All animals were sacrificed at 6 weeks postsurgery. A 2 cm segment of the spinal cord, centered on the injury site, was harvested and postfixed in 4% neutral buffered formalin for 48 h. Subsequently, all tissues underwent alcohol processing before being embedded in paraffin blocks. The sections were then cut into 5 μm thick slices and stained with hematoxylin and eosin to cover the entire injury site.

Immunofluorescence staining

For immunofluorescence staining of CD45, NeuN, and MFN2, the tissue slides were deparaffinized with xylene, hydrated by a graded ethanol solution, and treated with 0.2% Triton-X in PBS for 25 min and a protein-blocking reagent for 1 h. Added primary antibody of CD45 (50165365, Thermo Fisher Scientific Inc.), MFN2 (9482, Cell Signaling Technology) at 1:100 dilution and incubated for 48 h at 4°C, and then added the red-fluorescent conjugated secondary antibody (Invitrogen) at 1:500 dilution for 2 h at room temperature. The sections then added the primary antibody of NeuN (MAB377, Merck Millpore) at 1:500 dilution and incubated for overnight at 4°C, and then added the green-fluorescent conjugated secondary antibody (Invitrogen) at 1:500 dilution for 1 h at room temperature. Cells were counterstained with DAPI (Vector Laboratories) to detect nuclei. Images were analyzed under a florescence microscope (Carl Zeiss).

Immunohistochemistry staining

IHC staining was used with a horseradish peroxidase-diaminobenzidine (HRP-DAB) staining kit (R&D Inc.) as described previously34. The samples were performed from the spinal cord of Sham, SCI, ESWT, and ESWT3. Polyclonal or monoclonal antibodies against IL1-β, IL6, FGF1, FGF2, and FGFR from Santa Cruz, USA; pERK from Cell Signaling, USA; were used as the primary antibodies. The specific binding of the secondary antibodies to the primary antibodies was visualized by employing HRP to catalyze the enzymatic conversion of the chromogenic substrate 3,3’-DAB, resulting in the formation of a brown precipitate. Following mounting and clearing, the sections were cover-slipped and examined using a Zeiss fluorescence microscope (Zeiss). Images were analyzed using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring). The percentage of immunolabeled positive cells out of the total cells in each area were counted, and the average of each specimen was used as the results.

Statistical analysis

The statistical analysis of the data were performed using SPSS software (version 26.0, SPSS Inc.). Statistics were performed using one way ANOVA and Dunn-Bonferroni nonparametric comparison for post-hoc which the Kruskal–Wallis test was significant. The results were summarized as the mean±SD, and P<0.05, P<0.01, and P<0.001 were considered statistically significant.

Results

The study design and SCI rat model

In the current experiments, the study design was presented in Figure 1A. The experimental groups were divided into four groups such as Sham, SCI, ESWT, and ESWT3. In the experiments, we compared the effects of one treatment of ESWT and three treatments of ESWT for SCI rat and established the SCI rat model. SCI rat model was established by twice clip compressions with jaw pressure of 60 gravity on T9 spinal cord (Fig. 1B). The damage of the spinal cord was observed as indicated by the black arrow (Fig. 1C). The dosage of ESWT (0.13 mJ/mm2, 500 impulses, 4 Hz) was referenced as previous studies and our experiences.

The Basso, Beattie, and Bresnahan score of ESWT3 is better than ESWT for treatment of SCI rat

In addition, we measured the motor function of the SCI rats after treatments. In the Figure 1D (7 weeks), the hind legs of the rat dragged in the SCI and ESWT groups. However, in the ESWT3 group, some of the rats had their paws turned upwards. The BBB score was used to assess the locomotor function of the hindlimbs on a 21-point scale in Sham, SCI, ESWT, and ESWT3 groups from 2, 3, 5, and 7 weeks (Fig. 1E). The maximum of 21 points in the BBB score was in the Sham group and the BBB score was dropped near to zero after surgery (SCI group). The BBB score was improved in ESWT (P<0.05 and P<0.05) and ESWT3 (P<0.05 and P<0.01) as compared with the SCI group at 5 and 7 weeks and ESWT3 (P<0.05) was better than ESWT group at 7 weeks. The results indicated that ESWT could improve the locomotor function of SCI rat and three times treatment of ESWT3 was better than once treatment of ESWT.

The dosage-effect of shockwave treatment to improve motor function in SCI rat

Further, the CatWalk gait analysis was performed for behavioral tests in Sham, SCI, ESWT, and ESWT3 groups before rat sacrificed. Every run and the results of each group were showed in the Figure 3. In the SCI group, there was no print intensity (Fig. 2A) and print length (Fig. 2B) parameters in the hindlimbs (pink as right hind and green as left hind) compared to the Sham group after the injury. After shockwaves treatment, the print intensity and print length were improved in the ESWT and ESWT3 groups as compared with SCI as well as ESWT3 was better than the ESWT group.

Figure 3.

Figure 3

Open in a new tab

The histological structure, inflammation cytokines and immunocells measurement in the spinal cord injury after treatments. (A) The morphological observation is performed in the Sham, SCI, ESWT, and ESWT3 groups. The scale bar is 500 μm. The red arrow is indicated gray matter and yellow arrow is indicated white matter in the spinal cord. (B) The immunohistochemistry staining of IL1-β in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of IL1-β. (C) The immunohistochemistry staining of IL6 in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of IL6. The images are 200x magnification. (D) The immunohistochemistry staining of CD68-positive macrophages in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of CD68-positive macrophages. The images are 200x magnification scale bar is 100 μm. (E) The immunofluorescence staining of CD45-positive leukocyte in the Sham, SCI, ESWT, and ESWT groups. Red arrows: increased leukocyte infiltration. Scale bar: 100 μm. (F) The panel is the fluorescence intensity of CD45-positive leukocyte. The *** P<0.001, ** P<0.01, and * P<0.05 are compared with the SCI group. N=4. ESWT, extracorporeal shockwave therapy; ESWT3, three ESWT treatments; SCI, spinal cord injury.

Figure 2.

Figure 2

Open in a new tab

The graphical print view and data for each group by gait analysis from CatWalk. (A) The intensity of the run patterns. (B) Print view of the run pattern. (C) Run average speed. (D) Maximum variation. The *** P<0.001, ** P<0.01, and * P<0.05 are compared with the SCI group. The ## P<0.01 are compared between ESWT and ESWT3 groups. N=8. ESWT, extracorporeal shockwave therapy; ESWT3, three ESWT treatments; SCI, spinal cord injury.

The CatWalk gait parameters were measured, including run average speed and maximum variation in Sham, SCI, ESWT, and ESWT3 (Figs 2C and D). The run average speed and maximum variation were significantly improved in ESWT (P<0.05 and P<0.01) and ESWT3 (P<0.05 and P<0.001) than SCI. The improvement of the ESWT3 group was better than the ESWT group in run average speed (P<0.01) and maximum variation (P<0.01).

Shockwave treatment promoted the histological structure, anti-inflammation as well as improved neuron and mitochondria function in the injury spinal cord of the rat

In the pathological analysis of SCI rats after treatments, the gray (red arrow) and white (yellow arrow) matters were destroyed in the lesion center of the SCI group and repaired in the ESWT and ESWT3 groups, as well as the ESWT3 group, was better than ESWT group (Fig. 3A) showing from HE staining.

In addition, inflammatory markers, macrophages, leukocytes, and mitochondrial proteins were measured in the SCI group and treatment groups (Figs 3 and 4). The inflammatory markers IL-1β, IL-6 (Figs 3B and C), and macrophage (CD68, Fig. 3D) were increased in the SCI group, as well as leukocyte (CD45, Figs 3E and F) levels, while neuronal cells (NeuN) and the mitochondrial function marker mitofusin 2 (MFN2) were decreased (Fig. 4). In contrast, the treatment groups, ESWT (P<0.05, P<0.001, and P<0.05) and ESWT3 (P<0.01, P<0.001, and P<0.001), reduced IL-1β, IL-6, and macrophage (CD68) levels, increased leukocyte (CD45) levels (P<0.05, P<0.01), and enhanced the expression of NeuN (ESWT3, P<0.05) and MFN2 (ESWT3, P<0.05) in cells. Furthermore, the ESWT3 group exhibited superior effects compared to the ESWT group in promoting histological structure, reducing inflammation, and improving neuronal cell and mitochondrial function in SCI rats.

Figure 4.

Figure 4

Open in a new tab

The NeuN and MFN2 measurements in the spinal cord injury after treatments. (A) The immunofluorescence staining of NeuN and MFN2 in the Sham, SCI, ESWT, and ESWT3 groups. (B) The related NeuN positive cells. (C) The related MFN2 positive cells. The ** P<0.01 and * P<0.05 are compared with SCI group. N=8. ESWT, extracorporeal shockwave therapy; ESWT3, three ESWT treatments; ROI, region of interest; SCI, spinal cord injury. The bright field of the images is 50x magnification. The images of immunofluorescence staining is 1000x magnification.

Shockwave treatment modulated the expression of inflammation-induced FGF1, FGF2, their FGFR1 receptor and phosphorylation of ERK in SCI rat

Additionally, we know that FGF1 and FGF2 play dual roles in tissue repair and inflammation. Endogenous FGF1 and FGF2 are highly expressed in inflammatory tissues through the pERK pathway to induce fibrosis, where they stimulate the expression of cytokines. However, exogenous administration of FGF1 and FGF2 can assist in tissue regeneration. However, the homeostasis of FGF1 and FGF2 are important for the tissue repair. In the results, the expression levels of endogenous FGF1, FGF2, their FGFR1 receptor and pERK were highly elevated in the SCI group. These levels were reduced to those observed in the Sham group after treatments in the ESWT (P<0.01, P<0.001, P<0.01, P<0.001) and ESWT3 (P<0.001, P<0.001, P<0.01, and P<0.001) groups (Fig. 5). In addition, ESWT3 (P<0.05) was better than ESWT group to inhibit the activity of phosphorylation of ERK. The results indicated that ESWT protected the damaged spinal cord by modulating inflammation-induced FGF1, FGF2, and pERK signaling.

Figure 5.

Figure 5

Open in a new tab

The expression of inflammation induced FGF1, FGF2, and FGFR1 receptor in the spinal cord injury after treatments. (A) The immunohistochemistry staining of FGF1-positive cells in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of FGF1-positive cells. The images are 200x magnification. (B) The immunohistochemistry staining of FGF2-positive cells in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of FGF2-positive cells. The images are 200x magnification. (C) The immunohistochemistry staining of FGFR1-positive cells in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of FGFR1-positive cells. The images are 200x magnification. (D) The immunohistochemistry staining of pERK-positive cells in the Sham, SCI, ESWT, and ESWT groups. The right panel is the expressed percentage of pERK-positive cells. The *** P<0.001, ** P<0.01, and * P<0.05 are compared with the SCI group. # P<0.05 is compared with the ESWT group. N=8. ESWT, extracorporeal shockwave therapy; ESWT3, three ESWT treatments; SCI, spinal cord injury.

Discussion

In the study, we demonstrated the dose-dependent effects of low-energy ESWT on motor function recovery, tissue regeneration, anti-inflammatory responses, and mitochondrial function in a rat model of SCI. We found that three treatments of ESWT (ESWT3) were more effective than a single treatment (ESWT) in improving motor function, as evidenced by run patterns, run average speed, maximum variation, and the BBB score. Additionally, ESWT3 significantly reduced the inflammatory markers IL-1β, IL-6, and macrophages, while increasing the levels of leukocytes, NueN, and MFN2 compared to the SCI and ESWT groups. Furthermore, ESWT3 was superior in modulating inflammation-induced FGF1, FGF2, FGFR1, and pERK signaling to support tissue repair after SCI.

Motor function recovery

SCI leads to severe motor impairment, and restoring locomotor function is a critical challenge in SCI treatment35. Emerging treatment modalities such as ESWT have shown promise in promoting tissue repair and enhancing motor function recovery in animal model with SCI36. ESWT is a noninvasive therapeutic technique that utilizes low-energy shockwaves to stimulate tissue regeneration and repair37. It has been successfully used in various musculoskeletal conditions, such as tendinopathies and fractures, but its application in SCI is relatively novel10,38. The fundamental principle of ESWT involves the delivery of mechanical energy to the affected area, triggering a cascade of cellular responses that promote healing and tissue regeneration37. Our results demonstrated that ESWT, particularly with three treatments (ESWT3), improved motor function significantly compared to the SCI and ESWT groups, as shown in the BBB score and CatWalk gait analysis (Figs 1, 2 and 3A). The improved run patterns, average speed, and reduced variability in gait further indicate the effectiveness of repeated ESWT treatments. This finding aligns with the growing evidence that ESWT promotes neuroprotection and recovery in SCI. Recently, ESWT is used for spinal cord ischemia and is demonstrated with safety and feasibility in patients39. In addition, a clinical trial protocol of ESWT for acute traumatic SCI on motor and sensory function is reported and one section of ESWT is used for the treatment40. However, the optimal dosage of ESWT for human SCI patients is yet to be precisely determined, and further research is needed to establish standardized protocols.

Anti-inflammatory effects and mitochondrial protection

SCI triggers an intense inflammatory response that can exacerbate tissue damage. In our study, ESWT3 significantly reduced the levels of IL-1β and IL-6, key inflammatory cytokines, as well as macrophage infiltration in the injured spinal cord (Fig. 3). These results suggest that repeated ESWT treatments provide better control of inflammation, potentially preventing secondary injury. These results suggest that repeated ESWT treatments provide better control of inflammation, potentially preventing secondary injury. Accumulating evidence reveal that the intrinsic dynamicity of the mitochondria plays a central role in regulating the inflammatory response41. Here, we observed that SCI significantly reduced MFN2 expression, which is crucial for mitochondrial function. ESWT3 restored MFN2 levels, further supporting its role in reducing inflammation and protecting mitochondrial integrity (Fig. 4). This is consistent with previous findings that mitochondrial dysfunction exacerbates inflammation, and MFN2 plays a central role in maintaining mitochondrial dynamics during injury42. The recovery of MFN2 expression by ESWT3 indicates its potential to preserve mitochondrial function, which is essential for neuronal survival and tissue repair. It is possible that ESWT3 administration not only reduces inflammatory response but also facilitates mitochondrial transportation in axons by maintaining the level of MFN2.

Immune response and leukocyte infiltration

The immune response plays a crucial role in regulating various pathological processes and has a major impact on the prognosis of SCI43. In SCI, the immune response coordinates the activation of different immune cells and the release of immune factors. Injury can activate T cells and/or B cells, triggering an autoimmune response in the nervous system and maintaining their activation for an extended period of time44. During the repair phase, numerous cell adhesion molecules appear on the surface of activated T cells, facilitating their adhesion to vascular endothelial cells and enabling their entry into the central nervous system, which inhibits axonal death and promotes neuroprotection45. Our study demonstrated that ESWT3 increased leukocyte infiltration in the injured spinal cord compared to the SCI and ESWT groups (Fig. 3E). This increase in immune cell infiltration likely contributes to the enhanced regenerative capacity observed in the ESWT3 group. Leukocytes, particularly T and B cells, play an essential role in modulating inflammation and promoting neuroprotection46. The ability of ESWT to stimulate immune cell involvement in tissue repair highlights its therapeutic potential in modulating the immune response in SCI recovery. However, further studies are needed to explore whether ESWT promotes cell adhesion molecule expression, facilitating leukocyte infiltration.

Modulation of FGF1, FGF2, and FGFR1 signaling

Abnormal expressions of FGFs/FGFRs involve the progression of fibrosis formation47. Elevated expression of FGF1 and FGFR1 in idiopathic pulmonary fibrosis. However, FGF1 and FGF2 play dual roles in tissue repair and inflammation48. The homeostasis of FGF1 and FGF2 is crucial for inflammatory tissue response and repair. In various experimental models, these growth factors have been demonstrated to enhance angiogenesis, accelerate wound healing, and facilitate the regeneration of damaged tissues49. This regenerative effect is attributed to their ability to stimulate cell proliferation, differentiation, and survival in the context of tissue injury. Therefore, while endogenous FGF1 and FGF2 may contribute to fibrosis under inflammatory conditions, their therapeutic administration can potentially override these fibrotic pathways and promote the restoration of normal tissue architecture. In this study, SCI rats exhibited elevated levels of FGF1, FGF2, and their receptor FGFR1, which were reduced after ESWT treatment (Fig. 5). The modulation of these growth factors by ESWT suggests a mechanism through which ESWT may limit fibrosis and support tissue repair. The reduction of FGF1 and FGF2 expression and signaling in the ESWT and ESWT3 groups further underscores the role of shockwaves in balancing inflammation and promoting regeneration. The ability of ESWT to modulate these growth factors offers promising insights for its application in clinical settings.

Limitations

While our study shows promising results regarding low-energy ESWT for SCI recovery, several limitations exist. First, the rat model used may not fully replicate the complexity of human SCI, limiting the direct translational potential. Further studies in large animal models or clinical trials are necessary. Second, the optimal ESWT treatment regimen (frequency, intensity, and duration) remains undetermined, requiring further research to standardize protocols. Third, the 6-week observation period does not address the long-term effects of ESWT, including the potential for chronic complications such as scarring and fibrosis. Lastly, the molecular mechanisms by which ESWT modulates FGF1, FGF2, FGFR1, and pERK signaling require more investigation to clarify their roles in tissue repair.

Conclusion

In conclusion, our study demonstrates that repeated low-energy ESWT (ESWT3) is more effective than a single treatment in improving motor function, reducing inflammation, and promoting tissue repair in a rat model of SCI. ESWT3 also enhances mitochondrial function and modulates the expression of inflammation-related growth factors. These findings provide new insights into the therapeutic potential of ESWT for SCI treatment, suggesting that optimal dosing is crucial for maximizing its benefits. Future research is needed to explore the underlying mechanisms of ESWT and determine standardized protocols for clinical applications in SCI patients.

Ethical approval

This study was subjected to the approval of the Institutional Animal Care and Use Committee (IACUC) at KCGMH (Approval number: 2019120603).

Consent

Not applicable.

Source of funding

The funding sources were from Chang Gung Medical Foundation, Kaohsiung Chang Gung Memorial Hospital (CRRPG8J0031, CRRPG8J0032, and CRRPG8J0033).

Author contribution

C.-C.H.: funding acquisition, methodology, writing – original draft, and writing – review and editing; K.L.H.W.: methodology and writing – original draft; J.-M.P.: data curation, supervision, validation, and writing – original draft; Y.-N.W.: formal analysis, investigation, and writing – original draft; H.-T.C.: data curation, methodology, and validation; M.-S.L.: methodology and writing – original draft; J.-H.C.: conceptualization, investigation, methodology, supervision, validation, writing – original draft, and writing – review and editing.

Conflicts of interest disclosure

The authors declare no conflicts of interest.

Research registration unique identifying number (UIN)

Not applicable.

Guarantor

Chieh-Cheng Hsu.

Data availability statement

The dataset used and analyzed in this study were obtained from the corresponding authors upon reasonable request.

Provenance and peer review

Invited paper for the special issue ‘Shockwave treatment’ and the guest editor is Dr Kandiah Raveendran.

Acknowledgements

The authors are grateful to the Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, for supporting this work.

Footnotes

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Published online 24 October 2024

Contributor Information

Chieh-Cheng Hsu, Email: t1234@cgmh.org.tw.

Jei-Ming Peng, Email: r.jmpeng@gmail.com.

Yi-No Wu, Email: 133838@mail.fju.edu.tw.

Hou-Tsung Chen, Email: sendoh740105@gmail.com.

Meng-Shiou Lee, Email: leemengshiou@mail.cmu.edu.tw.

References

  • 1.Wang TY, Park C, Zhang H, et al. Management of acute traumatic spinal cord injury: a review of the literature. Front Surg 2021;8:698736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Li S, Dinh HTP, Matsuyama Y, et al. Molecular mechanisms in the vascular and nervous systems following traumatic spinal cord injury. Life 2022;13:9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zhang Y, Al Mamun A, Yuan Y, et al. Acute spinal cord injury: pathophysiology and pharmacological intervention (Review). Mol Med Rep 2021;23:417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hellenbrand DJ, Quinn CM, Piper ZJ, et al. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J Neuroinflammation 2021;18:284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Jaerve A, Muller HW. Chemokines in CNS injury and repair. Cell Tissue Res 2012;349:229–248. [DOI] [PubMed] [Google Scholar]
  • 6.Deumens R, Koopmans GC, Honig WM, et al. Chronically injured corticospinal axons do not cross large spinal lesion gaps after a multifactorial transplantation strategy using olfactory ensheathing cell/olfactory nerve fibroblast-biomatrix bridges. J Neurosci Res 2006;83:811–820. [DOI] [PubMed] [Google Scholar]
  • 7.Su H, Wu Y, Yuan Q, et al. Optimal time point for neuronal generation of transplanted neural progenitor cells in injured spinal cord following root avulsion. Cell Transplant 2011;20:167–176. [DOI] [PubMed] [Google Scholar]
  • 8.Fan B, Wei Z, Feng S. Progression in translational research on spinal cord injury based on microenvironment imbalance. Bone Res 2022;10:35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Orban M, Elsamanty M, Guo K, et al. A review of brain activity and EEG-based brain–computer interfaces for rehabilitation application. Bioengineering 2022;9:768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Auersperg V, Trieb K. Extracorporeal shock wave therapy: an update. EFORT Open Rev 2020;5:584–592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ikeda K, Tomita K, Takayama K. Application of extracorporeal shock wave on bone: preliminary report. J Trauma 1999;47:946–950. [DOI] [PubMed] [Google Scholar]
  • 12.Chen YJ, Wurtz T, Wang CJ, et al. Recruitment of mesenchymal stem cells and expression of TGF-beta 1 and VEGF in the early stage of shock wave-promoted bone regeneration of segmental defect in rats. J Orthopaed Res 2004;22:526–534. [DOI] [PubMed] [Google Scholar]
  • 13.Wang FS, Yang KD, Kuo YR, et al. Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect. Bone 2003;32:387–396. [DOI] [PubMed] [Google Scholar]
  • 14.Wang CJ. Extracorporeal shockwave therapy in musculoskeletal disorders. J Orthop Surg 2012;7:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wang CJ, Wang FS, Yang KD, et al. Shock wave therapy induces neovascularization at the tendon-bone junction. A study in rabbits. J Orthop Res 2003;21:984–989. [DOI] [PubMed] [Google Scholar]
  • 16.Wang CJ. An overview of shock wave therapy in musculoskeletal disorders. Chang Gung Med J 2003;26:220–232. [PubMed] [Google Scholar]
  • 17.Lee TC, Huang HY, Yang YL, et al. Vulnerability of the spinal cord to injury from extracorporeal shock waves in rabbits. J Clin Neurosci 2007;14:873–878. [DOI] [PubMed] [Google Scholar]
  • 18.Lee TC, Huang HY, Yang YL, et al. Application of extracorporeal shock wave treatment to enhance spinal fusion: a rabbit experiment. Surg Neurol 2008;70:129–134; discussion 34. [DOI] [PubMed] [Google Scholar]
  • 19.Lee TC, Yang YL, Chang NK, et al. Biomechanical testing of spinal fusion segments enhanced by extracorporeal shock wave treatment in rabbits. Chang Gung Med J 2009;32:276–282. [PubMed] [Google Scholar]
  • 20.Bolt DM, Burba DJ, Hubert JD, et al. Determination of functional and morphologic changes in palmar digital nerves after nonfocused extracorporeal shock wave treatment in horses. Am J Vet Res 2004;65:1714–1718. [DOI] [PubMed] [Google Scholar]
  • 21.Wang CJ, Huang HY, Yang K, et al. Pathomechanism of shock wave injuries on femoral artery, vein and nerve. An experimental study in dogs. Injury 2002;33:439–446. [DOI] [PubMed] [Google Scholar]
  • 22.Wu YH, Liang HW, Chen WS, et al. Electrophysiological and functional effects of shock waves on the sciatic nerve of rats. Ultrasound Med Biol 2008;34:1688–1696. [DOI] [PubMed] [Google Scholar]
  • 23.Hausner T, Pajer K, Halat G, et al. Improved rate of peripheral nerve regeneration induced by extracorporeal shock wave treatment in the rat. Exp Neurol 2012;236:363–370. [DOI] [PubMed] [Google Scholar]
  • 24.Hausner T, Nógrádi A. The use of shock waves in peripheral nerve regeneration: new perspectives? 2013;109:85–98. [DOI] [PubMed] [Google Scholar]
  • 25.Wu YH, Lun JJ, Chen WS, et al. The electrophysiological and functional effect of shock wave on peripheral nerves. Ann Int Conf IEEE Eng Med Biol Soc 2007;2007:2369–2372. [DOI] [PubMed] [Google Scholar]
  • 26.Takahashi N, Ohtori S, Saisu T, et al. Second application of low-energy shock waves has a cumulative effect on free nerve endings. Clin Orthop Relat Res 2006;443:315–319. [DOI] [PubMed] [Google Scholar]
  • 27.Ohtori S, Inoue G, Mannoji C, et al. Shock wave application to rat skin induces degeneration and reinnervation of sensory nerve fibres. Neurosci Lett 2001;315:57–60. [DOI] [PubMed] [Google Scholar]
  • 28.Murata R, Nakagawa K, Ohtori S, et al. The effects of radial shock waves on gene transfer in rabbit chondrocytes in vitro. Osteoarthritis Cartilage 2007;15:1275–1282. [DOI] [PubMed] [Google Scholar]
  • 29.Yamaya S, Ozawa H, Kanno H, et al. Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury. J Neurosurg 2014:1–12. [DOI] [PubMed] [Google Scholar]
  • 30.Kilkenny C, Browne WJ, Cuthill IC, et al. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010;8:e1000412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Christie SD, Comeau B, Myers T, et al. Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone. Neurosurg Focus 2008;25:E5. [DOI] [PubMed] [Google Scholar]
  • 32.Gollmann-Tepeköylü C, Nägele F, Graber M, et al. Shock waves promote spinal cord repair via TLR3. JCI Insight 2020;5:e134552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995;12:1–21. [DOI] [PubMed] [Google Scholar]
  • 34.Kuo YR, Wang CT, Wang FS, et al. Extracorporeal shock-wave therapy enhanced wound healing via increasing topical blood perfusion and tissue regeneration in a rat model of STZ-induced diabetes. Wound Repair Regen 2009;17:522–530. [DOI] [PubMed] [Google Scholar]
  • 35.Anjum A, Yazid MD, et al. Spinal cord injury: pathophysiology, multimolecular interactions, and underlying recovery mechanisms. Int J Mol Sci 2020;21:7533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Alavi SNR, Neishaboori AM, Yousefifard M. Extracorporeal shockwave therapy in spinal cord injury, early to advance to clinical trials? A systematic review and meta-analysis on animal studies. Neuroradiol J 2021;34:552–561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Simplicio CL, Purita J, Murrell W, et al. Extracorporeal shock wave therapy mechanisms in musculoskeletal regenerative medicine. J Clin Orthop Trauma 2020;11:S309–S318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Moya D, Ramón S, Schaden W, et al. The role of extracorporeal shockwave treatment in musculoskeletal disorders. J Bone Joint Surg 2018;100:251–263. [DOI] [PubMed] [Google Scholar]
  • 39.Graber M, Nägele F, Röhrs BT, et al. Prevention of oxidative damage in spinal cord ischemia upon aortic surgery: first‐in‐human results of shock wave therapy prove safety and feasibility. J Am Heart Assoc 2022;11:e026076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Leister I, Mittermayr R, Mattiassich G, et al. The effect of extracorporeal shock wave therapy in acute traumatic spinal cord injury on motor and sensory function within 6 months post-injury: a study protocol for a two-arm three-stage adaptive, prospective, multi-center, randomized, blinded, placebo-controlled clinical trial. Trials 2022;23:245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Missiroli S, Genovese I, Perrone M, et al. The role of mitochondria in inflammation: from cancer to neurodegenerative disorders. J Clin Med 2020;9:740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Misko AL, Sasaki Y, Tuck E, et al. Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration. J Neurosci 2012;32:4145–4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Ahmed A, Patil A-A, Agrawal DK. Immunobiology of spinal cord injuries and potential therapeutic approaches. Mol Cell Biochem 2017;441:181–189. [DOI] [PubMed] [Google Scholar]
  • 44.Jones TB. Lymphocytes and autoimmunity after spinal cord injury. Exp Neurol 2014;258:78–90. [DOI] [PubMed] [Google Scholar]
  • 45.Hu J-G, Shen L, Wang R, et al. Effects of Olig2-overexpressing neural stem cells and myelin basic protein-activated t cells on recovery from spinal cord injury. Neurotherapeutics 2011;9:422–445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Dwyer LJ, Maheshwari S, Levy E, et al. B cell treatment promotes a neuroprotective microenvironment after traumatic brain injury through reciprocal immunomodulation with infiltrating peripheral myeloid cells. J Neuroinflammation 2023;20:133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.MacKenzie B, Korfei M, Henneke I, et al. Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis. Respir Res 2015;16:83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Xie Y, Su N, Yang J, et al. FGF/FGFR signaling in health and disease. Signal Transduct Target Ther 2020;5:181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Farooq M, Khan AW, Kim MS, et al. The role of fibroblast growth factor (FGF) signaling in tissue repair and regeneration. Cells 2021;10:3242. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The dataset used and analyzed in this study were obtained from the corresponding authors upon reasonable request.

Articles from International Journal of Surgery (London, England) are provided here courtesy of Wolters Kluwer Health

RESOURCES