Abstract
Background
Existing animal models of myofascial pain syndrome (MPS) target different pathological mechanisms and often rely on invasive or intensive interventions to induce inflammatory or neurophysiological responses. However, models specifically addressing peripheral myofascial structural remodeling, particularly fibrosis-associated phenotypes, are still limited. Therefore, a simplified and reproducible model focusing on myofascial remodeling–related dysfunction is needed.
Methods
Fifty adult male Sprague–Dawley rats were randomly assigned to control, sham, one-, two-, or three-session groups (n = 10/group). Intervention groups received combined mechanical impact (right gastrocnemius, 2.5 g, 12 m/s) followed by downhill treadmill running (−16°, 16 m/min, 60 min) every five days, for one, two, or three cycles. Behavioral and functional outcomes, including muscle nodule incidence, mechanical pain threshold, ankle mobility, and balance beam performance, were assessed weekly for four weeks. Ultrasonography, muscle stiffness, and histology evaluated structural changes.
Results
Control and sham groups showed no alterations. All intervention groups developed transient nodules and reduced pain thresholds at week 1. One-session rats recovered by week 4, two-session rats partially recovered, while three-session rats exhibited persistent nodules, mechanical hyperalgesia, increased stiffness, and long-term functional impairment. Ultrasonography and histology in the three-session group revealed localized structural abnormalities, fiber disorganization, and collagen deposition.
Conclusion
Three sessions of mechanical impact combined with eccentric exercise at five-day intervals produced a reproducible rat model of repetitive muscle injury–induced MPS-like features. The model consistently induced peripheral myofascial structural remodeling, mechanical hyperalgesia, increased muscle stiffness, and functional deficits for at least four weeks. This time-efficient protocol may provide a useful platform for mechanistic studies and preclinical evaluation of therapies targeting peripheral fibrotic and mechanical mechanisms associated with myofascial pain. However, it primarily reproduces peripheral myofascial alterations rather than the full clinical and pathophysiological spectrum of MPS.
Keywords: myofascial pain syndrome, animal model, mechanical impact, eccentric exercise, muscle nodules, collagen deposition, muscle stiffness
Introduction
Myofascial pain syndrome (MPS) is a common cause of chronic musculoskeletal pain, imposing significant clinical burden, yet its pathophysiology remains incompletely understood.1–3
Several hypotheses have been proposed to explain the development of MPS. Early theories primarily focused on abnormal motor endplate activity, including spontaneous electrical activity (SEA) and endplate noise (EPN), as potential neuromuscular sources of trigger points.4,5 Subsequent models, such as the energy crisis theory, emphasized the role of sustained muscle contraction, local ischemia, and metabolic stress in maintaining peripheral nociceptive input.6–9
More recently, MPS has been increasingly conceptualized as a myofascial disorder involving not only neuromuscular dysfunction but also alterations in the extracellular matrix and tissue biomechanics.10–15 In this framework, repetitive mechanical overload, micro-injury, local ischemia, and neuro-immune interactions are thought to contribute to extracellular matrix remodeling, increased tissue stiffness, and persistent sensitization.10,13–15 Importantly, although electrophysiological abnormalities have been widely investigated in experimental settings, they are not included in routine clinical diagnostic criteria, which rely primarily on palpable muscle abnormalities and pressure-evoked pain.1,16–19
Animal models are essential for elucidating the pathophysiological mechanisms of MPS and for evaluating therapeutic interventions. Although various experimental approaches have been developed, each presents specific limitations. Acidic saline models primarily induce localized inflammation, but the underlying pathogenic mechanism does not reflect the common etiological factors observed in clinical MPS. Some artificial electrical stimulation or stretching protocols mainly reflect acute contraction-related injury and assess outcomes over a short, acute period, failing to capture the chronic, focal, and progressive nature of MPS. Blunt trauma models mainly focus on inducing spontaneous electrical activity (SEA) rather than a fibrosis-dominant phenotype, and therefore often require repeated mechanical impacts, which may be excessively severe and introduce additional nonspecific tissue damage.20–23 Collectively, these limitations highlight the need for a simplified, time-efficient, and reproducible animal model that better reflects the chronic, focal, and structurally remodeling features of MPS, particularly those driven by mechanical overload and fibrosis.
The present study aimed to establish a rat model of repetitive muscle injury–induced MPS-like phenotypes by combining localized mechanical impact with eccentric exercise. Rather than attempting to reproduce the full clinical spectrum of MPS, this model was specifically designed to reflect a mechanically induced, fibrosis-dominant phenotype characterized by peripheral myofascial remodeling, mechanical hyperalgesia, and functional impairment, while placing less emphasis on electrophysiological outcomes.
Materials and Methods
Animals
Adult male Sprague–Dawley rats (6 weeks old, 220–250 g) were obtained from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (SCXKLu2022-0006, Shandong, China). Animals were housed under standard laboratory conditions (22 ± 2 °C, 12-h light/dark cycle) with free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital (QFSYYPZ2023012301). Animal care and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. This study was reported in accordance with the ARRIVE guidelines. Detailed descriptions of anesthesia and euthanasia methods are provided in the corresponding sections.
Randomization and Blinding
Animals were randomly assigned to experimental groups using a computer-generated randomization sequence generated by an investigator not involved in the interventions, outcome assessments, or data analysis. Group allocation was concealed in sequentially numbered, opaque sealed envelopes and was revealed only after animal enrollment. Investigators responsible for behavioral assessments, muscle nodule evaluation, ultrasound imaging, histological analyses, and statistical analyses were blinded to group allocation throughout the study. Due to the nature of the intervention procedures, the investigator performing the mechanical impact and eccentric exercise protocols could not be blinded but was not involved in outcome assessment or data analysis.
Experimental Design
A total of 50 rats were randomly assigned into five groups (n = 10 per group): control, sham, one-session, two-session, and three-session groups. The control group received no intervention throughout the experimental period. The sham group underwent the same anesthesia, fixation, and handling procedures as the intervention groups but without mechanical impact or eccentric exercise, to control for the effects of experimental manipulation. Rats in the intervention groups received combined mechanical impact followed by eccentric exercise once every five days. According to group allocation, animals received one, two, or three intervention cycles, respectively. Each intervention cycle consisted of a single mechanical impact session followed by one session of downhill treadmill running performed on the following day. All animals were subsequently monitored and assessed weekly for four weeks after the final intervention. An a priori sample size calculation was not performed. Group sizes were selected based on previous studies employing similar experimental paradigms and were consistent with commonly used sample sizes in comparable MPS animal studies.
Acclimation and Preconditioning
During the acclimation period, rats in the sham and intervention groups underwent adaptive downhill treadmill running for three consecutive days to minimize exercise-related stress. Each session lasted 15 minutes at a speed of 16 m/min with a downhill incline of 16° (−16°) using a motor-driven treadmill. The treadmill was equipped with a mild electrical stimulation grid at the rear end to encourage continuous running. No mechanical impact was applied during the acclimation period.
Mechanical Impact Procedure
Mechanical impact was delivered to the right gastrocnemius muscle using a customized slingshot device. The projectile consisted of a cylindrical wooden rod with a diameter of 6 mm and a mass of 2.5 g. The initial velocity of the projectile was calibrated to 12 m/s, corresponding to a kinetic energy of 0.822 J.24 Projectile velocity was verified prior to experiments using repeated calibration measurements to ensure stable impact energy across experimental sessions.
During the mechanical impact procedure, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). After achieving adequate anesthesia, animals were positioned in a standardized supine posture with the right hind limb fixed, and the hip, knee, and ankle joints flexed at approximately 90° to ensure consistent exposure of the gastrocnemius muscle belly. To minimize inter-animal variability, the same anatomical landmark—the midpoint of the medial gastrocnemius muscle belly—was identified in every animal before each intervention. Briefly, the medial aspects of the knee and ankle joints were identified by palpation and connected by a reference longitudinal line along the medial shank. A transverse line perpendicular to this longitudinal axis was then defined at its midpoint, corresponding to the mid-shank level. Along this transverse line, the medial border of the tibia and the posterior contour of the calf were identified by palpation, and the midpoint between these two landmarks was determined. This point, corresponding to the most prominent region of the medial calf, was defined as the midpoint of the medial gastrocnemius muscle belly and was consistently used as the standardized site for mechanical impact throughout the study.
Mechanical impact was applied vertically to the mid-belly of the right gastrocnemius muscle using a customized slingshot device. The projectile was released from a fixed distance and orientation, perpendicular to the muscle surface, to standardize the direction and magnitude of force delivery. All procedures were performed by the same trained investigator to reduce operator-dependent variability. This standardized positioning was used for all animals to ensure consistency of the impact site across intervention cycles.
This protocol was designed to induce localized mechanical microtrauma without causing overt muscle rupture or skeletal injury. Animals were monitored after each impact session to confirm the absence of gross muscle rupture, skin damage, or fracture, thereby ensuring consistency of injury severity among animals.
Eccentric Exercise Protocol
Eccentric exercise was performed using a motor-driven animal treadmill (KT-PW animal treadmill, Kaerwen, China). Downhill running was achieved by setting the treadmill at a decline angle of 16°, indicating a downward slope of 16°. Rats ran at a speed of 16 m/min for a duration of 60 minutes per session. To ensure continuous running, a mild electrical stimulus was applied at the rear of the treadmill when necessary.
Eccentric exercise was initiated on the second day following each mechanical impact procedure and was performed once per intervention cycle. One complete intervention cycle (session) consisted of a single mechanical impact followed by one bout of eccentric exercise on the subsequent day.
A three-day recovery period without any intervention was allowed between consecutive intervention cycles. According to group allocation, rats in the one-session, two-session, and three-session groups underwent one, two, or three complete intervention cycles, respectively. No eccentric exercise was performed in the control or sham groups.
This delayed loading protocol was designed to mimic repetitive mechanical microtrauma followed by muscle activation under conditions of incomplete recovery.
Assessment of Muscle Nodules
Palpable muscle nodules or taut bands in the gastrocnemius muscle were evaluated weekly throughout the observation period by two independent investigators blinded to group allocation. Muscle nodules were defined as localized, firm regions within the muscle belly that elicited withdrawal, vocalization, or avoidance behavior upon manual compression. Only nodules independently identified and confirmed by both examiners were recorded. When a muscle nodule was identified, the corresponding location on the skin surface was marked to ensure consistent reassessment of the same site during subsequent evaluations.
To reduce subjective bias, assessments were performed according to predefined criteria, and only consensus findings between the two blinded investigators were included in the analysis.
Mechanical Pain Threshold Assessment
Mechanical pain threshold of the gastrocnemius muscle was assessed using a pressure algometer (ZP-10N, AILIGU, China) equipped with a circular probe (tip diameter: 8 mm). Rats were gently restrained in a standardized position, and the probe was applied perpendicularly to the identified muscle nodules on the modeled (right) side.
Pressure was increased at a constant rate of 1 N/s until the rat exhibited a clear nociceptive response, defined as hind limb withdrawal, paw licking, or avoidance behavior. The peak pressure value at the moment of response was recorded as the mechanical pain threshold. Each measurement was repeated three times with an interval of at least 30 seconds, and the mean value was used for subsequent analysis. All assessments were performed by an investigator blinded to group allocation.
Ankle Joint Range of Motion
Ankle joint range of motion (ROM) was assessed to evaluate functional impairment of the hindlimb. Rats were gently restrained in a supine position, with the knee joint stabilized to prevent compensatory movement. Passive dorsiflexion and plantarflexion of the right ankle joint were performed manually until a firm end point was reached without causing discomfort.
A standard goniometer was aligned with the lateral malleolus as the axis of rotation, with one arm parallel to the fibula and the other aligned with the fifth metatarsal bone. The maximal dorsiflexion and plantarflexion angles were recorded, and the total ankle ROM was calculated as the sum of both angles.
Each measurement was repeated three times, and the average value was used for subsequent analysis. All assessments were performed by the same investigator blinded to group allocation to minimize measurement bias.
Balance Beam Walking Test
The balance beam walking test was conducted to assess motor coordination and functional performance of the hindlimbs. The apparatus consisted of a wooden beam measuring 100 cm in length and 1.5 cm in width, elevated 30 cm above the floor. A escape platform was placed at one end of the beam to encourage traversal.
Prior to formal testing, all rats were trained to walk across the beam for three consecutive days to familiarize them with the apparatus and minimize learning-related variability.
During testing, each rat was placed at the starting end of the beam and allowed to walk freely toward the escape platform. The time required to traverse the beam was recorded using a stopwatch as the primary outcome measure.
Each rat completed three trials with adequate rest between trials, and the average traversal time was used for subsequent analysis. All assessments were performed by an investigator blinded to group allocation.
Ultrasound Assessment
Ultrasonographic examination of the gastrocnemius muscle was performed using a high-resolution small-animal ultrasound system (Vevo 3100, FUJIFILM VisualSonics, Canada) equipped with a linear array transducer operating at a center frequency of 40 MHz.
Rats were anesthetized and positioned in supine posture. The right hind limb was gently secured to maintain a consistent and natural extension. The skin overlying the marked muscle nodule site was shaved and coated with acoustic coupling gel to ensure optimal signal transmission. The transducer was placed longitudinally and transversely over the gastrocnemius muscle with minimal pressure to avoid tissue deformation.
B-mode imaging was used to acquire real-time images of muscle architecture. Imaging parameters, including imaging depth (12 mm), gain, and focal zone, were kept constant for all animals throughout the experiment to ensure consistency.
Structural abnormalities were identified as localized heterogeneous echogenic regions, characterized by hyperechoic foci surrounded by hypoechoic areas, compared with the homogeneous echo pattern observed in normal muscle tissue.
All ultrasound examinations were performed by the same experienced operator blinded to group allocation.
Muscle Stiffness Measurement
Muscle stiffness was assessed using a controlled compression method. A standardized force of 0.2 N was applied perpendicularly to the muscle surface, and the resulting indentation depth of the muscle tissue was recorded. Muscle stiffness was quantified based on indentation depth, with smaller indentation values indicating increased muscle stiffness.
All ultrasonographic and stiffness measurements were performed at the same marked site for each animal and were conducted by a single experienced operator blinded to group allocation.
Histological Analysis
At the end of week 4, rats were deeply anesthetized and euthanized by an overdose of sodium pentobarbital administered intraperitoneally at a dose of 150 mg/kg. Death was confirmed by cessation of heartbeat and respiration before tissue collection. Gastrocnemius muscles were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin staining was used to assess muscle fiber morphology and inflammatory infiltration. Masson’s trichrome staining was performed to evaluate collagen deposition and fibrotic changes.
Statistical Analysis
Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 27.0 (IBM Corp., Armonk, NY, USA), and graphs were generated using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene’s tests, respectively. For repeated measurements over time, two-way repeated-measures ANOVA was used to evaluate the effects of group, time, and their interaction. When the assumption of sphericity was violated, Greenhouse–Geisser correction was applied. For comparisons among multiple groups at a single time point or endpoint measurement, one-way ANOVA was used. Dunnett’s multiple comparisons test was applied as the post hoc test to compare each experimental group with the control group. A two-sided P value < 0.05 was considered statistically significant.
Results
Establishment of a Reproducible Model of MPS-Like Featuresthrough Repeated Mechanical Impact Combined with Eccentric Exercise
To investigate the effects of repeated mechanical impact combined with eccentric exercise on the development of myofascial pain syndrome–like features, rats were subjected to one, two, or three intervention cycles and dynamically observed for four weeks (Figure 1). Behavioral, functional, imaging, and histological assessments were conducted to comprehensively evaluate pain sensitivity, muscle nodules, functional impairment, and tissue remodeling.
Figure 1.
Experimental timeline and intervention protocol. Experimental timeline and modeling protocol for myofascial pain syndrome. All rats were acclimated to the housing environment for one week prior to experimentation. Rats assigned to the intervention groups underwent adaptive downhill (eccentric) treadmill exercise for three consecutive days (A) before the first mechanical impact. Each intervention cycle consisted of a single mechanical impact applied to the gastrocnemius muscle, followed by downhill treadmill exercise 24 h later (B). Intervention cycles were performed once every five days and repeated one, two, or three times depending on group allocation. Behavioral and functional assessments were conducted weekly during the four-week observation period following the final intervention (C). At the end of week 4, ultrasonographic imaging was performed, followed by collection of gastrocnemius muscle tissues for histological analysis (D).
Repeated Intervention Cycles Induced Progressive Formation of Muscle Nodules, Mechanical Hyperalgesia, and Increased Muscle Stiffness
Palpable muscle nodules were not observed in either the control or sham groups throughout the entire observation period. In contrast, all intervention groups developed muscle nodules to varying extents following mechanical impact combined with eccentric exercise (Figure 2A). However, the persistence of these nodules differed among groups. In the one-session (1-s) and two-session (2-s) groups, muscle nodules gradually diminished and eventually disappeared during the follow-up period. Notably, only rats in the three-session (3-s) group exhibited sustained and stable muscle nodules that persisted throughout the four-week observation period.
Figure 2.
Muscle nodule formation, mechanical pain sensitivity, and muscle stiffness. Muscle nodule incidence, mechanical pain threshold, and stiffness of the gastrocnemius muscle following repeated intervention cycles. (A) Incidence of palpable muscle nodules in each experimental group during the observation period. (B) Mechanical pain threshold measured at the marked muscle nodule site using a pressure algometer. (C) Muscle stiffness quantified by indentation depth under a standardized compressive force of 0.2 N, with smaller indentation depth indicating increased muscle stiffness. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group; ##P < 0.01,###P < 0.001 compared with the sham group.
Mechanical pain threshold remained stable in the control and sham groups throughout the four-week observation period. In contrast, all intervention groups exhibited a marked decrease in mechanical pain threshold at week 1 following the combined mechanical impact and eccentric exercise, indicating the development of mechanical hyperalgesia (Figure 2B). During the subsequent observation period, divergent recovery patterns were observed among the intervention groups. In the one-session (1-s) group, pain threshold gradually increased and returned to levels comparable to the control group by week 4. The two-session (2-s) group also demonstrated a progressive increase in pain threshold over time; however, values at week 4 remained significantly lower than those of the control and sham groups. Notably, the three-session (3-s) group exhibited persistently reduced pain threshold throughout the four-week period, with consistently significant differences compared with both control and sham groups.
Muscle stiffness, as assessed by indentation depth under standardized compression, remained unchanged in the control and sham groups during the observation period (Figure 2C). In the intervention groups, no significant alteration in muscle stiffness was observed in the one-session (1-s) group compared with the control and sham groups. In contrast, both the two-session (2-s) and three-session (3-s) groups exhibited significantly reduced indentation depth, indicating increased muscle stiffness. Notably, the increase in stiffness was more pronounced in the 3-s group than in the 2-s group, suggesting a session-dependent exacerbation of mechanical tissue alterations.
Repeated Mechanical Impact and Eccentric Exercise Impaired Ankle Joint Mobility and Motor Coordination
Functional assessment revealed an acute reduction in ankle joint mobility in all intervention groups at week 1 following the repeated mechanical impact and eccentric exercise (Figure 3A). Passive ankle dorsiflexion range of motion remained stable in the control and sham groups throughout the observation period. In contrast, rats in the one-session (1-s) and two-session (2-s) groups exhibited a gradual recovery of joint mobility over time, with no significant difference compared with the control group by week 4. Although partial recovery was also observed in the three-session (3-s) group, dorsiflexion range of motion stabilized at weeks 3 and 4 and remained significantly lower than that of the control and sham groups, indicating persistent functional impairment.
Figure 3.
Ankle joint mobility and motor coordination. Effects of repeated mechanical impact and eccentric exercise on ankle joint range of motion and motor performance. (A) Passive ankle dorsiflexion range of motion measured over the four-week observation period. (B) Balance beam walking performance assessed by traversal time. Data are presented as mean ± SD. ***P < 0.001 compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the sham group.
Similarly, motor coordination assessed by the balance beam walking test demonstrated a significant increase in traversal time in all intervention groups at week 1 compared with the control and sham groups (Figure 3B). Rats in the one-session (1-s) and two-session (2-s) groups exhibited a gradual improvement in motor performance over the subsequent weeks, with traversal times returning to levels comparable to the control group by week 4. In contrast, although partial recovery was observed in the three-session (3-s) group, traversal time remained prolonged at weeks 2–4 and was significantly higher than that of the control and sham groups, indicating persistent impairment in motor coordination.
Ultrasonographic Imaging Revealed Localized Structural Abnormalities in the Gastrocnemius Muscle Following Repeated Interventions
To further characterize structural changes associated with muscle nodule formation, ultrasonographic evaluation was performed at week 4. Representative longitudinal and transverse images demonstrated homogeneous muscle architecture in the control group, whereas the three-session (3-s) group exhibited localized regions characterized by a central hyperechoic area surrounded by hypoechoic zones, corresponding to the palpated muscle nodules (Figure 4A).
Figure 4.
Ultrasonographic assessment of gastrocnemius muscle structural alterations at week 4. Ultrasonographic comparison of gastrocnemius muscle structure between the control group and the three-session (3-s) intervention group at the end of the four-week observation period. (A) Representative longitudinal and transverse ultrasound images of the gastrocnemius muscle from the control group and the three-session group, demonstrating structural differences at the muscle nodule site. (B) Enlarged views of the corresponding muscle nodule regions in the three-session group highlighting localized structural abnormalities. Arrows indicate regions corresponding to palpable muscle nodules.
Magnified views of the affected regions revealed disrupted muscle fiber organization and focal structural irregularities in the 3-s group (Figure 4B). These ultrasonographic abnormalities were spatially consistent with the marked muscle nodule sites, supporting the presence of localized pathological alterations following repeated intervention cycles.
Repeated Interventions Induced Muscle Atrophy, Cellular Alterations, and Collagen Deposition in the Gastrocnemius Muscle
Gross examination of isolated gastrocnemius muscles revealed distinct morphological alterations only in the three-session (3-s) group (Figure 5A). Muscles from the 3-s group appeared visibly smaller in volume, with an uneven surface texture and a pale yellowish coloration, suggestive of tissue degeneration and atrophy. In contrast, gastrocnemius muscles from the control, sham, one-session, and two-session groups maintained a plump appearance with smooth surfaces and normal reddish coloration.
Figure 5.
Gross morphology, muscle mass, cellular alterations, and collagen deposition in the gastrocnemius muscle. Gross appearance, muscle weight, histological changes, and quantitative analysis of fibrotic remodeling in the gastrocnemius muscle following repeated mechanical impact and eccentric exercise. (A) Representative gross images of isolated gastrocnemius muscles from each experimental group. (B) Quantification of gastrocnemius muscle weight in each group. (C) Representative hematoxylin and eosin–stained sections showing muscle fiber morphology and cellular infiltration. (D) Quantitative comparison of nuclei number per field based on H and E-stained sections. (E) Representative Masson-stained sections illustrating collagen fiber deposition. (F) Quantitative analysis of collagen fiber area fraction (collagen volume fraction, CVF) in the gastrocnemius muscle. Data are presented as mean ± SD. *P < 0.05, ***P < 0.001 compared with the control group. #P < 0.05, ###P < 0.001 compared with the sham group.
Quantitative analysis of muscle weight showed a significant reduction in the 3-s group compared with the control and sham groups (Figure 5B). No significant differences in muscle mass were observed among the control, sham, one-session, and two-session groups.
Histological examination with hematoxylin and eosin staining demonstrated well-organized muscle fibers with uniform morphology and minimal cellular infiltration in the control, sham, and one-session groups (Figure 5C). In the two-session (2-s) group, muscle fibers largely preserved their overall architecture but exhibited mild interstitial expansion accompanied by increased infiltration of inflammatory cells and scattered fibroblasts, indicating early pathological remodeling. In contrast, the three-session (3-s) group showed pronounced histopathological alterations, including markedly disorganized muscle fiber arrangement, substantial variation in fiber diameter, and extensive infiltration of inflammatory cells and fibroblasts within the interstitial spaces, suggesting advanced tissue injury and fibrotic progression.
Quantitative analysis revealed a significant increase in nuclei number per field in the 2-s and 3-s groups compared with the control and sham groups, with the highest nuclei density observed in the 3-s group (Figure 5D).
Masson staining revealed minimal collagen fiber deposition in the control and sham groups, indicating preserved normal muscle architecture (Figure 5E). In the one-session (1-s) and two-session (2-s) groups, mild increases in interstitial collagen fibers were observed, primarily distributed around muscle fascicles and within the perimysial regions, suggesting early fibrotic remodeling. In contrast, the three-session (3-s) group exhibited marked accumulation of collagen fibers with dense and extensive deposition throughout the interstitial spaces, indicating pronounced fibrosis of the gastrocnemius muscle.
Quantitative analysis demonstrated that the collagen volume fraction (CVF) was significantly increased in the 1-s, 2-s, and 3-s groups compared with the control and sham groups, with a progressive elevation corresponding to the number of intervention cycles. The highest CVF values were observed in the 3-s group (Figure 5F).
Three Intervention Cycles Were Sufficient to Induce Stable Myofascial Pain Syndrome–Like Pathological and Functional Features
Collectively, repeated mechanical impact combined with eccentric exercise induced a spectrum of MPS-like features, including persistent muscle nodules, mechanical hyperalgesia, increased muscle stiffness, functional impairment, localized structural abnormalities, and fibrotic tissue remodeling. These effects exhibited a clear dependence on the number of intervention cycles.
Notably, three intervention cycles were sufficient to consistently produce stable behavioral, functional, imaging, and histological alterations, suggesting that the three-session protocol represents an efficient and reproducible method for modeling muscle injury–related pain and tissue remodeling.
Discussion
This study evaluated the effects of different repetition frequencies of mechanical impact combined with eccentric exercise on the development of myofascial pain syndrome (MPS)–like changes in rats. The results indicate that three intervention sessions consistently induced mechanical hyperalgesia, functional impairment, persistent muscle nodules, localized ultrasonographic abnormalities, and characteristic histopathological remodeling. Together, these findings suggest that this protocol is sufficient to reproduce key pathological and functional features associated with MPS, particularly those related to peripheral myofascial remodeling, tissue stiffness, and pain sensitivity. However, the present findings should not be interpreted as reproducing all pathophysiological components of the syndrome.
Previous experimental models of MPS have generally relied on repeated mechanical stimulation or invasive procedures, particularly those based on the motor endplate hypothesis.7,13,20,21 In these models, electrophysiological abnormalities such as spontaneous electrical activity were often considered essential indicators of trigger point formation, requiring frequent or prolonged interventions to achieve stable changes.20–23,25–28 Although such approaches have provided insight into neuromuscular mechanisms, their complexity and extended experimental duration may limit practicality in some research settings. Other commonly used models, including acidic saline injection and artificial electrical stimulation protocols, reproduce distinct aspects of MPS pathophysiology. Acidic saline models predominantly induce nociceptive sensitization through inflammatory mechanisms, whereas electrical stimulation models emphasize contraction-related muscle injury and neuromuscular dysfunction. These approaches collectively highlight the multifactorial nature of MPS and suggest that no single experimental model fully captures all components of the disorder.
In clinical practice, however, the diagnosis of MPS is primarily based on the presence of palpable muscle nodules or taut bands associated with localized tenderness and movement restriction, without routine reliance on electrophysiological assessments.1,17,29,30 Although neurophysiological validation remains important, a model centered on clinically observable peripheral manifestations may still provide practical value for studying selected aspects of MPS.
The present model was developed with an emphasis on myofascial structural remodeling, including extracellular matrix accumulation, collagen deposition, altered tissue stiffness, and localized disruption of muscle architecture. The progressive histological changes observed in the two-session and three-session groups, together with the marked fibrotic features in the three-session group, support the involvement of tissue remodeling processes in persistent pain and localized tissue abnormalities. This interpretation is consistent with one of several proposed frameworks for MPS pathogenesis, namely the fibrosis/extracellular matrix remodeling hypothesis. However, alternative mechanisms, including motor endplate dysfunction, the energy crisis hypothesis, peripheral sensitization, and central sensitization, have also been implicated in MPS development. Because these mechanisms were not directly assessed in the present study, the current findings should be interpreted primarily as evidence of mechanically induced myofascial remodeling rather than a comprehensive representation of all MPS-related processes. In addition, the ultrasonographic findings of localized heterogeneous echo patterns further reflect focal structural abnormalities within the muscle. These observations are consistent with increasing evidence that myofascial trigger points involve localized fibrotic and biomechanical alterations rather than solely neuromuscular dysfunction.14,31–38
Within this framework, three repeated mechanical insults combined with eccentric exercise were sufficient to produce sustained pathological changes resembling those seen in MPS. While one-session and two-session interventions induced transient pain sensitivity and mild structural alterations, only the three-session protocol resulted in persistent nodules, increased stiffness, functional deficits, and pronounced fibrosis. These findings suggest that repeated mechanical loading beyond a certain threshold is required to induce stable myofascial remodeling. Within the scope of the present study, this threshold appears sufficient to generate a reproducible fibrosis-dominant MPS-like phenotype.
An important feature of this model is the reduced number of intervention cycles needed to achieve stable pathological outcomes. Compared with previously reported protocols requiring numerous stimulations, the current approach shortens experimental time and limits unnecessary tissue injury, while still reproducing selected peripheral features associated with MPS.21,23 This makes the model suitable for mechanistic studies focusing on extracellular matrix remodeling, fibrosis progression, and changes in tissue mechanical properties, as well as for evaluating therapeutic strategies aimed at modulating myofascial pathology. Importantly, the present model is intended to represent a peripheral structural component of MPS rather than the entire syndrome. Its greatest utility may therefore lie in investigating the relationship between repetitive mechanical loading, tissue remodeling, fibrosis, and pain-related behaviors.
Several limitations of the present study should be acknowledged. First, although the model reproduced several MPS-like behavioral, functional, imaging, and histological features, neurophysiological mechanisms were not investigated. Electrophysiological characteristics such as spontaneous electrical activity, motor endplate dysfunction, peripheral sensitization, and central sensitization were not assessed. Consequently, the present model should be interpreted as reflecting primarily a peripheral tissue-remodeling phenotype rather than the full spectrum of MPS pathophysiology. Second, although palpable nodules and localized ultrasonographic abnormalities were consistently observed, their equivalence to clinically defined myofascial trigger points remains uncertain. Additional validation using electrophysiological, molecular, or imaging approaches is required. Third, the observation period was limited to four weeks following the final intervention. While stable fibrotic remodeling and functional impairment were observed within this timeframe, longer-term studies are warranted to determine the persistence or progression of these pathological changes. Finally, the present model focused on the gastrocnemius muscle in male rats, and further validation in other muscle groups and in female animals may improve the generalizability of the findings.
Conclusion
In conclusion, repeated mechanical impact combined with eccentric exercise provides a time-efficient and reproducible rat model that reproduces selected peripheral structural and functional features associated with MPS. The severity and persistence of pathological and functional alterations exhibited a clear dependence on the number of intervention cycles. While one or two sessions primarily induced transient and partially reversible changes, three intervention cycles represented the minimal and sufficient condition to consistently produce stable muscle nodules, persistent mechanical hyperalgesia, increased muscle stiffness, functional impairment, localized structural abnormalities, and progressive fibrotic remodeling of the gastrocnemius muscle.
This simplified three-session protocol balances pathological relevance with experimental feasibility, minimizing experimental burden while reliably reproducing a fibrosis-dominant and mechanically driven MPS-like phenotype characterized by tissue remodeling, altered mechanical properties, and pain-related behavioral changes. Because neurophysiological mechanisms, central sensitization, and electrophysiological characteristics of trigger points were not evaluated, the present model should be interpreted primarily as representing peripheral myofascial remodeling rather than the full spectrum of MPS pathophysiology. Furthermore, the relationship between the observed muscle nodules and clinically defined myofascial trigger points remains to be further validated. The model provides a practical experimental platform for investigating myofascial remodeling, collagen deposition, and biomechanical alterations, and may be useful for preclinical evaluation of therapeutic strategies targeting the fibrotic and mechanical mechanisms associated with myofascial pain. Future studies incorporating neurophysiological and central nervous system assessments will be important to further define the relationship between peripheral tissue remodeling and the broader pathogenesis of MPS.
Funding Statement
This study was supported by the Shandong Provincial Natural Science Foundation, China (grant no. ZR2023MH118); the Mount Taishan Scholar Distinguished Expert Project, China (grant no. tstp20231253); the National Administration of Traditional Chinese Medicine Science and Technology Project, China (grant no. GZY-KJS-2023-019) and the National Natural Science Cultivation Foundation of China (grant no. QYPY2022NSFC0609).
Disclosure
Zixu Lv and Wenlong Yang are co-first authors for this study. The authors report no conflicts of interest in this work.
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