Purpose: Few studies have assessed the effects of sphingosine kinase 1/sphingosine-1-phosphate (SPHK1/S1P) on microangiogenesis at rat myofascial trigger points (MTrPs) using contrast-enhanced ultrasonography (CEUS). This study aimed to address these deficiencies. Here, we investigated the effects of SPHK1/S1P on MTrP microangiogenesis and the value of CEUS in evaluating these effects.
Methods: Forty Sprague‒Dawley rats were subdivided into two groups: control and MTrP groups. MTrPs were established by 8 weeks of the strike procedure combined with eccentric motion and 4 weeks of recovery. All rats were euthanized after having undergone CEUS with an overdose of pentobarbital sodium. MTrP and control tissue samples were removed for haematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) imaging. The tissue was dehydrated, cleared, and embedded before sectioning. The sections were then incubated overnight at 4°C, and immunohistochemistry was carried out with primary antibodies including rabbit anti-CD31, rabbit anti-SPHK1and rabbit anti-S1PR1.
Results: MTrP rats exhibited spontaneous electrical activity (SEA) and a local twitch response (LTR) during electromyography (EMG) examination. The CEUS time-intensity curves (TICs) showed that the perfusion intensity in the MTrPs and surrounding tissue area was increased, with faster perfusion than in normal sites, while the TICs in the control group slowly increased and then slowly decreased. The correlation coefficient between the microvessel density (MVD) and sphingosine 1-phosphate receptor 1 (S1PR1) was 0.716 (p < 0.01). Spearman correlation analysis revealed that Spearman’s rho (ρ) values between the MVD and peak intensity (PI), between the MVD and area under the curve (AUC), and between the MVD and SPHK1 were > 0.5 (p < 0.05), > 0.7 (p < 0.01), and > 0.7 (p < 0.01), respectively.
Conclusion: CEUS is valuable for detecting microangiogenesis within MTrPs, and SPHK1/S1P plays an important role in promoting MTrP tissue microangiogenesis.

Keywords: ultrasound, myofascial pain syndrome, diagnosis, angiogenesis