Stellate ganglion block (SGB) is a method with a broad range of applications for the prevention and treatment of chronic diseases, including the treatment of pain (Gunduz and Kenis-Coskun, 2017; Ueshima, 2019), the treatment of sleep disturbance after breast cancer (Jin et al., 2018), and complementary therapy between chronic ulcerative colitis and hemodialysis (Gunduz and Kenis-Coskun, 2017; Jin et al., 2018). An efficient and stable animal model is particularly important for the further study of SGB. The anatomical structure and function of the stellate nerve are similar in rats and humans (Abdi and Yang, 2005). The stellate ganglion, located at the level of the 2nd thoracic vertebra, is formed by the fusion of the inferior cervical ganglion and the first three thoracic sympathetic ganglia (C6-T3). Due to the opposite relationship of the dominant components, vagal blockade may cause failure of the expected therapeutic effect of SGB to meet expectations. The therapeutic mechanism is mainly associated with sympathetic nerve block (Filippiadis and Kelekis, 2016). The anatomical structure of the target area can be clearly revealed by ultrasound, and the needle, the catheter and drug diffusion can also be shown simultaneously. Therefore, ultrasound effectively increases the success rate of target nerve block and reduces complications (Liu et al., 2016; Kanna et al., 2019a). Ultrasound guided stellate ganglion block has been widely validated in many clinical trials of human subjects (Shibata et al., 2007; Gofeld et al., 2009; Aleanakian et al., 2020). In clinical trials, it has been confirmed that ultrasound guided stellate ganglion block can greatly reduce adverse reactions and complications related to blind injection, such as intravascular injection, hematoma formation, temporary paralysis of recurrent laryngeal nerve and esophageal injury (Lee et al., 2012; Narouze, 2012). However, ultrasound-guided SGB in rats has not yet been reported. This study aims to establish the methodology of ultrasound-guided rat SGB and we propose a clearly stated hypothesis that ultrasound-guided rat SGB is significantly better than traditional blind SGB. A new murine model for ultrasound-guided SGB was established in this study. This study provides a reference for the accurate preparation of animal models of SGB.

This study initiated a new method for SGB in rats and verified its accuracy. Under ultrasound guidance, the operative duration, minimum effective volume of local anesthetic, and incidence of brachial plexus block, vagal block, respiratory depression and death were significantly reduced. These results confirm the reliability and safety of this technique.

Ultrasound-guided SGB or sympathetic trunk block in humans is generally performed in the neck (Baek et al., 2018; Singh et al., 2021). The human sympathetic trunk and the vagus nerve are located in different fascial layers (Yuan et al., 2017; Freitas et al., 2020). The long cervical muscle can be exhibited by ultrasound at the C6-7 level. Local anesthetic drugs could be injected on the surface to achieve SGB and perfectly evade the vagus nerve, achieving the effect of a simple sympathetic block. A clinical retrospective case study found that ultrasound guided stellate ganglion block was safe and effective in reducing sympathetic maintenance pain in patients with CRPS and neuropathic pain syndrome, and confirmed that the incidence of complications (3.9% hoarseness, 3.4% dysphagia, 0.6% hematoma) was lower than that of non-ultrasound guided techniques (Aleanakian et al., 2020). However, unlike humans, the rat neck sympathetic and vagus nerves are parallel in the same vascular sheath (Bookout and Gautron, 2021). Preliminary experimental results indicate that neck block probably blocks also the vagus nerve. Therefore, the first intercostal space was chosen as the target for SGB in this study. In this gap, the stellate ganglion is far from the vagus nerve and brachial plexus. Low-dose local anesthetics achieve a pure sympathetic nerve block and avoid brachial plexus block.

Traditional SGB in rats relies on body surface markers and the blind injection of drugs. Without sufficient experience, the probability of accidental injury of nerves or blood vessels is greatly increased, and the blockade effect is unsatisfactory (Filippiadis and Kelekis, 2016). The effect of traditional SGB will inevitably be affected by anatomical variations in rats, and an undesirable nerve blockade effect can be determined by the brachial plexus stimulation sign alone. Besides, various methods have been used to localize the stellate ganglion, such as CT (Christie and Martinez, 1995), MRI (Hogan and Erickson, 1992; Slappendel et al., 1995), and fluoroscopy (Elias, 2000; Abdi et al., 2004; Maki et al., 2016). Fluoroscopy-guided stellate ganglion block has been widely reported as a new technology (Maki et al., 2016). It shows the position of the needle and the diffusion range of drugs in real time through radiography, which is more suitable for providing diagnostic evaluation and has been proven effective in human clinical research (Elias, 2000). However, in animal experiments, fluoroscopy-guided stellate ganglion block needs to be carried out in a laboratory environment that can effectively resist radiation, such as a lead plate, which has high requirements for the laboratory, and also has the harm of radiation to laboratory personnel. Under the guidance of ultrasound, the stellate ganglion block of animals has no radiation, high repeatability, and can also visualize the blocking process and drug diffusion, so it is more feasible to use ultrasound to create models in animal experiments. Ultrasound technology clearly depicts the anatomical structures of muscles, intervertebral foramina, nerve roots, and blood vessels in the target area, thereby mediating navigation of the puncture needle to the pre-determined position to increase the nerve block success rate (Liu et al., 2016; Kanna et al., 2019b). In this study, the volume of drugs used was smaller and the operative duration was shorter in the ultrasound-guided group, indicating that the method is more stable and easier to master.

The anatomical exploration revealed that the brachial plexus ganglion was located at the upper edge of the first rib near the clavicle, while the stellate ganglion is located at the lower edge of the first rib. Brachial plexus irritation indicates the successful injection of drugs for SGB, which is contrary to the characteristics of the anatomical structure. It also reveals that the capacity required to achieve the effect of SGB is often larger than expected. The reason why this indication has been widely used may be the failure to find other positioning methods better than the blind probing method. Ultrasound guidance solves this problem and provides a better way to accurately establish the model.

Hemorrhage, nerve palsy, and pneumothorax are serious complications of SGB, which will affect the results of observation and research in experimental models (Okuda et al., 2016; Hirota et al., 2017). There were two cases of hematoma and two cases of pneumothorax in the blind detection group. This is attributed to the fact that the blind detection operation lacks sufficient precision in control of the needle tip and requires repeated puncture to induce brachial plexus irritation, which causes damage to blood vessels, pleura and nerves (Saranteas et al., 2019).

A systematic review of animal studies showed that the harmful effects of nerve block are mostly caused by mechanical damage, volumetric pressure and neurotoxicity (Sondekoppam and Tsui, 2017; Hewson et al., 2018; Barrington and Lirk, 2019). In this study, the minimum effective volume for rat SGB under ultrasound guidance was 0.020 ml, while the minimum effective volume for rat SGB under blind detection was 0.639 ml. Ultrasound guidance can effectively avoid the harmful effect of volumetric pressure and achieve better therapeutic effects with smaller volumes. Most nerve injuries are caused by mechanical stimulation (direct puncture trauma, pressure injury) or neurotoxic chemical factors (Sondekoppam and Tsui, 2017). The degree of nerve injury during nerve block depends on the angle and size of the puncture. Under visual conditions, the position of the needle, the angle of needle insertion and the diffusion of the drug are clearly displayed to pilot the puncture needle to the pre-determined position, minimizing the occurrence of mechanical injury (Gadsden et al., 2014). We found that within the 95% CI of the lowest effective volume, the complication rate of the ultrasound-guided method was significantly lower than that of the blind detection method. Methylene blue was mixed with 1.5% lidocaine to ascertain the exact range of lidocaine diffusion during ultrasound-guided SGB. The results confirmed that the lidocaine injected under ultrasound guidance was accurately distributed near the stellate ganglion without spreading to the brachial plexus. While ensuring the effect of SGB, brachial plexus block should be avoided as much as possible. In terms of the operative duration, the ultrasound-guided positioning method was significantly faster than the blind detection method, which has notable advantages. For the operator, it is easier to establish a rat SGB model under ultrasound guidance, and the model is more stable.

It is worth noting that there are still some shortcomings in this study. First, although ultrasound guidance technology was used, it could not provide an accurate image of the stellate ganglia. Thus, the method still depends on the positioning of the adjacent ganglia, blood vessels and bone tissues. This defect needs to be resolved by higher-resolution ultrasound. Second, the up-and-down sequential method of Dixon was used to investigate the minimum effective volume and ED95. However, this method is not sufficiently accurate to estimate the ED95. In further research, the partial coin sequential method will be used. Third, ultrasound-guided SGB surgery requires high-frequency ultrasound equipment; thus, this method is not as portable as the blind detection method. Fourth, due to the small number of experimental animals, it was difficult to compare the incidence of complications of SGB with ultrasound guidance and blind detection in terms of hemothorax and pneumothorax.

Ultrasound-guided SGB allowed visualization of the block process, and the effect of the block was definite. The SGB model established in this manner is more stable, avoiding complications to the greatest extent. Compared with traditional techniques, this method can be better applied in SGB-related research.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This animal study was reviewed and approved by Animal Experimentation Committee of Fujian Medical University (No: FJMU IACUC 2020-0063).

KZ and X-DW: carried out the studies and modify the manuscript. S-ZL and LC: data collection, statistical analysis, repeated reading of the manuscript, and management of the author’s team. Y-JT, C-JZ, and PK: data collection, organization of data collection, and statistical analysis. M-NC: statistical analysis, methodological contributions, and repeated reading of the manuscript. H-XW and YC: data collection and discussion of the manuscript. L-CQ: repeated reading of the manuscript. All authors contributed to the article and approved the submitted version.