Assessment of the effects of transforming growth factor beta1 (TGF-β1)-Smad2/3 on fibrosis in rat myofascial trigger points using point shear wave elastography

Experimental animals and grouping

Forty 5-week-old specific pathogen-free Sprague‒Dawley rats (healthy males) were purchased from the Animal Experimental Center of Guangxi Medical University (Certificate of Conformity: SCXK Gui2009-0002) and randomly divided into a control group (n = 10) and an MTrPs group (n = 30).The groups of animals were housed separately at a density of three animals per cage. The environment was controlled, with a humidity of 50–70%, a temperature of 22–24 °C, and a 12/12 h light/dark cycle.

MTrPs model rats

There is general agreement that muscle overuse or direct trauma to the muscle can lead to the development of MTrPs (Money, 2017). The MTrPs model was established as described by Huang et al. (2013) via a blunt strike in combination with eccentric exercise (Fig. 1) for 8 weeks. In this study, the right vastus medialis muscle was selected as the modelling area, and the remaining procedure was the same as that described in the reference literature. The model was considered successful on the basis of the following criteria: the right vastus medialis muscle exhibited one or more tense belts and contracture nodules on palpation, a local twitch response (LTR) was produced by insertion of an electrode needle into the nodules, high-frequency spontaneous electrical activity (SEA) was observed on electromyography (EMG) under resting conditions, and a continuous spontaneous potential occurred for more than 30 s. The region was marked when two or more of the above criteria were consistent with MTrPs.

Paw withdrawal threshold assessment

The paw withdrawal threshold was measured with von Frey filaments (Nahm et al., 2017) at 2, 4, 6 and 8 weeks of modelling and at 2 and 4 weeks after the completion of modelling. The weight of each animal was measured between 13:00 and 18:00 PM. The temperature of the test environment was controlled at between 24 °C and 26 °C, the humidity was maintained between 50% and 70%, and the environment was kept quiet. Increasing stimulation pressure was manually applied with von Frey filaments of increasing stiffness (2, 4, 6, 8, 10, 15, 26 g) in the centre of the ball of the right foot in the rats. The filament stiffness was recorded when evidence of the paw withdrawal reflex occurred, such as lifting, withdrawing, swinging or licking. The test was repeated five times for each rat with an interval of at least 2 min. The maximum and minimum values were removed, and the average of three tests was calculated as the paw withdrawal threshold (g).

Electromyographic examination

Electromyography (EMG) was performed after the modelling was completed. All rats were intraperitoneally anaesthetized with sodium pentobarbital (50 mg/kg of body weight) and subjected to hair removal. The right lower limb region of each rat was completely exposed. The rats were then attached to a retainer. EMG equipment (Keypoint9033A07, Dendi EMG, Denmark) was used at a sampling frequency of 20 HZ–10,000 Hz, sensitivity of 0.2 mv/grid, and scanning speed of 10 ms/grid. Then, electroacupuncture electrodes(Φ0.3 mm, 30 gauge) were inserted into the significantly strained muscle belts to detect the LTR and SEA, including fibrillation potential (I/II), positive sharp waves, and endplate spines. When the above SEA appeared with specific rhythmic waves on the electromyograph, images were recorded for 2 min. The rats in the control group were evaluated by EMG at the same position. All the above procedures were performed by an EMG physician with 8 years of experience.

Ultrasound image processing

After the EMG examination, all rats were then subjected to ultrasound using the Mindray ResonaI9 Clinical Ultrasound (US) system (Mindray Biomedical Electronics Co., Ltd, Shenzhen, China) and a linear array transducer (L9-3 s) probe (3.6–9.0 MHz). The probe was gently placed on the marks for scanning, and the MTrPs in the right vastus medialis muscle were observed in multiple sections to avoid anisotropy artefacts. Then, using point shear wave elastography to measure the shear wave velocity (SWV) of MTrPs, a range of 3 mm × 3 mm was selected as the region of interest (ROI). The average value of three measurements was calculated for statistical analysis. The data are expressed in m/s. The same location was selected and measured in the control group. The above procedures were performed by two diagnostic sonographers with 8 and 10 years of experience.

Haematoxylin and eosin (H&E) staining procedure

All rats were euthanized after point shear wave elastography examination and given an overdose of chloral hydrate, and the MTrPs tissues were perfused with paraformaldehyde (4%) through the heart for histological examination. The muscle with MTrPs was removed and fixed with 4% paraformaldehyde overnight. The tissues were subsequently embedded in paraffin and sectioned into 4 µm thick sections. Next, the sections were deparaffinized in xylene and rehydrated through a graded alcohol solution. Finally, the sections were stained with H&E and observed under a microscope at different magnifications (Eclipse Ci-L, Nikon, Toyko, Japan).

Masson staining procedure

The MTrPs tissue sections were dewaxed and dehydrated, stained with haematoxylin for 5 min, washed with water and subjected to counterstaining for 5 min. The sections were stained with acid fuchsin solution for 5 min, washed with weak acid working solution for 1 min, and washed with phosphomolybdic acid solution for 1 min. The samples were then subjected to aniline blue staining for 1 min, washing with weak acid working solution for 1 min, gradient ethanol dehydration, and clearing, followed by sealing with neutral gum. The relationship between the blue collagen-positive area and the total tissue area was evaluated by microscopic observation. The collagen volume fraction was analysed and calculated with Aipathwell image analysis software (collagen volume fraction = collagen area in the visual field/measured visual field area).

Transmission electron microscopy

MTrPs tissue blocks 1 mm * 2 mm * 3 mm in size were fixed in 2.5% glutaraldehyde phosphate buffer for 2 h. The samples were fixed, dehydrated, soaked, embedded, and cured according to typical electron microscopy preparation procedures. Thin slices subjected to 3% acetic acid uranium and lead nitrate staining were observed by transmission electron microscopy (TEM) (HT7800/HT7700, Hitachi, Tokyo, Japan) and images of 30 randomly selected fields of view were captured.

Immunohistochemistry

MTrPs tissues from each group were sliced at a thickness of 4 mm. In turn, the slices were placed into dewaxing solution, dehydrated in an alcohol gradient, and incubated in 3% hydrogen peroxide solution for 25 min at room temperature while protected from light. The tissues were blocked by dropping 3% BSA solution onto the sample for 30 min and incubated with rabbit anti-TGF-β1 (1:200, WL02193, Wanleibio, China), and rabbit anti-P-SMAD2/P-SMAD3 (1:200, WL02305, Wanleibio, China) primary antibodies at 4 °C overnight. The tissue was then covered with a secondary antibody (HRP labelled) of the same species as the primary antibody. DAB was used for colour development, the nuclei were restained, and the tissues were dehydrated for sealing and microscopic examination. The nuclei stained with haematoxylin were blue, and positive expression of DAB was indicated by light yellow to tan staining.

Western blotting

MTrPs tissues were collected and lysed in RIPA buffer (Solarbio, Beiijng, China). Afterwards, a BCA protein assay kit was used to quantify the protein. Western blotting (WB) was carried out as previously described (Lv et al., 2020) with the following antibodies: rabbit anti-TGF-β1 (1:1,000,WL02193;Wanleibio, Beijing, China), and rabbit anti-P-SMAD2/P-SMAD3 (1:1,000, WL02305, Wanleibio, Beijing, China). Adobe Photoshop CS2 was used for target protein band cutting, and Image-Pro Plus was used to analyse the grey values of the protein bands.

Statistical analysis

SPSS version 23.0 (Chicago, IL, USA) was used for data analysis, and GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to plot the data. All data are expressed as the mean (95% CI of the mean) or median (IQR). An independent sample T test was used to analyse the differences between groups, and the Mann‒Whitney U test was used for nonnormally distributed variables. The correlations were determined by calculating Pearson linear correlation coefficients or Spearman rank correlation coefficients. Statistical significance was accepted at p < 0.05.

Animal model results

Of the 30 MTrPs rats, 15 rats were excluded from the experiment due to death from anaesthesia overdose, fracture, or substandard palpation and EMG examination. Therefore, 25 rats were included in the final analysis (MTrPs group: n = 15, control group: n = 10). A detailed description of the modelling is provided in Table 1.

EMG results

The MTrPs were identified with EMG after 4 weeks of rest. Only a horizontal baseline appeared in the control group, while there was more SEA, as indicated by fibrillation potential (II) and the presence of positive sharp waves in the MTrPs group; in addition, increased numbers of endplate spines were observed in the MTrPs group (Fig. 1D, Table 2).

Ultrasound STQ findings

Two-dimensional ultrasound examination of the MTrPs group (Figs. 2A and 2C) revealed abnormalities in or near tense belt regions. The main manifestations included uneven echo or low echo, increased blood flow and greater SWV. In the control group (Figs. 2B and 2D), there were no abnormalities on ultrasonography, only muscle fibres with a uniform echo and no blood flow.

Comparison of pathological morphology and collagen volume fraction between the control group and the MTrPs group

H&E staining. In the MTrPs group, muscle cell disarrangement, fibrous connective tissue hyperplasia, neovascularization, inflammatory cell infiltration and nuclei in the interior or centre of the cells were observed (Fig. 3A). In the control group, the muscle cells were closely arranged, uniformly coloured, and clearly demarcated. Nuclei were located at the edge, and several muscle cells were clustered into bundles (Fig. 3B).

Masson staining. In the MTrPs group, red muscle fibres were disordered and squeezed, collagen fibre deposition increased significantly, and the fibres were thick and wrapped around new muscle cells and intermuscular membranes (Fig. 3C, yellow arrow). In the control group (Fig. 3D), the muscle fibres were arranged neatly, and the ratio of muscle fibres to collagen fibres was moderate.

Comparison of skeletal muscle ultrastructure between the control group and the MTrPs group

Compared with the control group, the MTrPs group exhibited a disordered arrangement of muscle fibres, shorter sarcomeres (P < 0.05) (Fig. 4D),and a twisted and broken Z-line. The number of mitochondria was decreased (P < 0.0001) (Fig. 4E), the double-membrane structure was blurred, and the sarcoplasmic reticulum was blurred in the MTrPs group. In the control group (Fig. 4C), the fibres of the vastus medialis muscle had an orderly arrangement, the myofilaments were evenly distributed, the length of the sarcomeres was uniform, and the Z line was clear and continuous. Mitochondria were evenly distributed among the myofibrils near the Z line, in an oval or long strip shape, with a normal size, morphology and structure and a visible bilayer membrane structure.

Comparison of TGF-β1 and p-Smad2/3 expression between the control group and the MTrPs group

TGF-β1 and p-Smad2/3 in tissue were yellow‒brown. The mean optical densities of TGF-β1 and p-Smad2/3 in the MTrPs group (0.011 ± 0.002 and 0.010 ± 0.002, respectively) were higher than those in the control group (0.009 ± 0.001 and 0.009 ± 0.001, respectively) (p < 0.05) (Figs. 5F and 5G).

In the MTrPs group, the collagen volume fraction was linearly correlated with SWV and TGFβ1 (IOD/area)

For the MTrPs rats, the SWV was 3.61 ± 0.33 m/s, which was higher than that of the control rats (2.37 ± 0.20 m/s) (p < 0.0001; MTrPs group: n = 15, control group: n = 10) (Fig. 6A). No significant correlation was observed between SWV and rat weight/ paw withdrawal threshold (p > 0.05) (Figs. 6B and 6C). In the MTrPs group, the collagen volume fraction was linearly correlated with SWV. The correlation coefficient between the collagen volume fraction and SWV was 0.539 (p < 0.05) (Fig. 6D). The correlation coefficient between the collagen volume fraction and TGF-β1 expression was 0.661 (p < 0.01) (Fig. 6E). No significant correlation was observed between the collagen volume fraction and p-Smad2/3 expression (p > 0.05) (Fig. 6F).