Silver Needle Thermal Therapy Relieves Pain, Repairs the Damaged Myofascial Fiber, and Reduces the Expression of 5-HT3 Receptors in the Spinal Cord of Rats with Myofascial Pain Syndrome
Yuanxin Huang, Xianglong Lv, Jing Yao, Wei Lu, Zilong Yu, Yue Qin, Yue Wang, Zhongjie Zhang, Cheng Yu, Lin Wang, Chunxin Wo
Pain Department, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, P. R, China
Correspondence Address:
Chunxin Wo
Pain Department, Affiliated Hospital of Guizhou Medical University, No. 28, Guiyi Street, Liuguangmen, Guiyang 550004, Guizhou Province
China
Lin Wang
Pain Department, Affiliated Hospital of Guizhou Medical University, No. 28, Guiyi Street, Liuguangmen, Guiyang 550004, Guizhou Province
China
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/0028-3886.360917
Background: There is an urgent clinical need to provide a theoretical basis for silver needle thermal therapy to Myofacial pain syndrome (MPS).
Objective: This study was conducted to explore the effect of silver needle thermal therapy on myofascial pain syndrome in rats.
Methods: MPS rat models were duplicated, and the rats were subsequently divided into model and treatment groups. A normal control group was synchronously set up. No treatment was given to the model group, whereas silver needle thermal therapy was administered to the treatment group. The thermal and mechanical pain threshold, the morphological structure as well as the expression of 5-HT3 receptors in the spinal cord were observed.
Results: Rats from the treatment group presented with a significantly higher pain threshold compared to the untreated model group. The myofascial arrangement of the affected part of the model group was disordered, and some muscle fibers were atrophied and deformed. Meanwhile, the myofascial arrangement of the treatment group became more regular than that of the model group. The expression levels of 5-HT3 receptor in the spinal cord of the untreated model group were significantly increased, while being markedly decreased in the treatment group.
Conclusions: Silver needle thermal therapy can augment the pain threshold of rats with MPS, repair the damaged myofascial membrane in the rats, and further reduce the expression of 5-HT3 receptors in the spinal cord of the MPS rats.
Keywords: 5-HT3 receptors, myofascial pain syndrome, pain threshold, silver needle thermal therapy, trigger point
Key Messages: MPS is a painful condition of myofascial trigger points (MTrPs) in skeletal muscles. Silver needle thermal therapy augments pain thresholdof rats with MPS as well as down regulates the expression of 5-HT3 receptors in the spinal cord and repair the damaged myofascial membrane, leading to pain control.
Myofascial pain syndrome (MPS) is a widespread and significant clinical problem characterized by localized muscle tenderness and pain, accounting for 15% of general medical visits. To be more precise, MPS is a painful condition of myofascial trigger points (MTrPs) in skeletal muscles and can occur alone or in tandem with other pain generators. Inherently, MTrPs serve as focal areas of taut bands in skeletal muscles that are hypersensitive to palpation. Application of manual pressure over an MTrP elicits a distinct local and referred pain that is consistent with the patient's presenting pain symptoms.[1] Overall, with varying ranges of pain, MPS exerts a significant impact on the quality of life of patients.[2]
Moreover, MPS presents with complicated pathogenesis such that both central and peripheral mechanisms are associated with the development of MPS. Peripheral strain on the muscles from sustained muscle tension habits, repetitive strain, and injuries contribute to localized progressive increases in oxidative metabolism and depleted energy supply. Furthermore, phasic modulation of excitatory and inhibitory interneurons supplied by high-threshold sensory afferents is also correlated with MPS, ramping up peripheral sensitization.[3] Meanwhile, the continual bombardment of primary afferent activity over time is known to lead to abnormal function and structural changes in the dorsal root ganglia and dorsal horn neurons. These changes are collectively known as central sensitization, which is clinically manifested in the form of allodynia, hyperalgesia, temporal summation of pain, and expansion of the receptive field of pain.[4] Nevertheless, the dysfunctional processes of MTrPs and the peripheral changes associated with the pain are still not fully understood.
Nociceptive input to the dorsal horn of the spinal cord is modulated by descending inhibitory and facilitatory control. Interestingly, the descending 5-hydroxytryptamine (5-HT) pathways are known to exert an inhibitory (descending inhibition) or facilitatory (descending facilitation) influence on the spinal processing of nociceptive information.[5],[6],[7] More recently, various animal studies have highlighted nerve injury–induced increased descending facilitation from the rostral ventromedial medulla (RVM) as an underlying mechanism of hypersensitivity.[8],[9],[10] At this conjecture, the exact roles of 5-HT receptors involved in pain in the spinal cord remain elusive. However, our esteemed peers have indicated the involvement of the 5-HT3 receptors (5-HT3R) in the spinal cord in descending facilitation. Furthermore, intrathecal infusion of the selective 5-HT3R antagonist, ondansetron, has been shown to reduce neuronal responses to mechanically punctuate stimuli and relieve hyperalgesia.[11]
The topic of our focus—silver needle thermal therapy—finds its origins in the Nine Needles of traditional Chinese medicine. It originated from the silver acupuncture therapy of Shanghai Lu's Traumatology Department and is regarded as a treatment modality under the guidance of traditional Chinese medicine theories of “selecting acupoints along the meridian”.[23] Since the 1970s, a large number of MPS patients in China have chosen to undergo silver needle thermal therapy and have achieved extremely satisfactory results, with a curative rate of over 90%. This can be attributed to the fact that silver needle thermal therapy presents various advantages over ordinary stainless steel acupuncture needles for the treatment of MPS. For instance, silver needle thermal therapy acts on the attachment point (area) of the myofascial membrane on the bone and exudes gentle heat at about 43°C instead of the general acupuncture concept which involves acupuncture points, thereby allowing deeper and wider acupuncture with silver needles. Additionally, silver needles with silver as the main raw material have a soft texture. This feature indicates that the needle can be pushed along the concave surface of the periosteum without breaking, which is conducive to acupuncture at a longer distance and enlarges the treatment surface and can be easily and accurately punctured to the trigger point. Moreover, the needle body of silver needles is relatively thick (1.0–1.1 mm in diameter) and prevents the needle from being broken or stuck due to excessive muscle contraction. Ordinary acupuncture needles made of stainless steel have a small diameter and a hard texture. If the needle is inserted into the deep tissue, and the needle is broken or stuck due to a strong muscle contraction reaction, it is fairly easy to cause accidents. Additionally, the high silver content of the silver needles allows the conduction of heat energy to deep diseased tissues for therapeutic effects.[12]
Although silver needle thermal therapy exhibits good clinical effects in the treatment of MPS, there are very few related studies, and a further dearth of studies focusing on its underlying mechanism in animal models. Clinical studies clearly provide a strong basis for the efficacy of silver needle thermal therapy in patients with chronic nonspecific low back pain (CNSLBP). Silver needle thermal therapy alleviated pain, improved function, and regulated autonomic nervous system activity for a long time without serious complications. The researchers suggest that the use of silver needle thermal therapy in the clinic should be promoted and it could be popularized in clinical practice.[13] Although we have found its excellent efficacy in clinical practice, there are only a few reports. There is still no relevant research on the specific biological mechanism of silver needle heat conduction therapy with better curative effect. Therefore, we took the first step to explore the mechanism in experimental animals and provide a more theoretical basis for its clinical application. In lieu of this, the current study set out to establish MPS rat models and treat the MPS rats with silver needle thermal therapy. Following silver needle thermal therapy (SNT), we documented the pain threshold, the morphological structure of affected myofascial membrane, and the expression patterns of 5-HT3 receptors in the spinal cord of the rats in an effort to elucidate and confirm the curative effect of silver needle thermal therapy with the help of MPS rat models.
MethodsAnimals and ethical statement
Adult Sprague–Dawley male rats (weighing 200–250 g; our university Animal Center, Guiyang, China) were included in the current study. All the selected rats were reared under the same conditions with similar body weights. The procured rats were individually housed in a temperature-controlled room, with natural light and relative humidity of 40%–60%. Animal experiments were carried out according to the guidelines of laboratory animal care, and experimentation protocols were approved by the Institutional Animal Care and Use Committee of our university. Extensive efforts were made to minimize the number and suffering of the experimental animals.
Experimental design
The rats were randomly divided into three groups using a web-based random number generator (Graph-Pad software) (n = 8 in each group): the normal control (NRC) group, the MPS model (MDC) group, and the silver needle thermal therapy (SNT) group. The rats in the NRC group were fed and maintained in the same environment as the other groups of rats without any intervention and were sacrificed at the same time as the other groups. Rat models of chronic MPS were established in the MDC group. After the models were successfully established, rats in the SNT group were subjected to silver needle thermal therapy.
MPS model
The model was established with reference to a previously described method.[14],[15] The process of MPS model establishment was divided into the intervention period and the recovery period. The intervention period lasted eight weeks and the recovery period lasted four weeks. Three days before the experiment, all rats were initially acclimated to using a provided treadmill (WI32812 multi-channel running, Dongxiyi Technology Co., Ltd., Beijing, China) for 15 min and accustomed to locomotion on the treadmill.
Following acclimatization, the rats were bluntly struck on the first day of the week during the intervention period. Briefly, the rats were anesthetized with an injection of 3 ml/kg of 1% sodium pentobarbital solution (Shanghai New Asiatic Pharmaceuticals Co., Ltd, Shanghai, China) into the abdominal cavity. After analgesia, the rats were fixed at the bottom of the striking device and a wooden stick on the striker was dropped from a height of 20 cm with a kinetic energy of 2.352 J onto the right medial femoral muscle. Centrifugal treadmill exercise was carried out on the second day, and the small animal treadmill was set to − 16° downhill running mode with a running speed of 16 m/min. All the rats in the MDC and SNT groups were subjected to training on the centrifugal treadmill.
During the recovery period, no intervention was administered to the rats. All the rats were allowed to continue their normal daily routines with routine feeding for four weeks.
Silver needle thermal therapy
Abdominal anesthesia was administered to the rats with 3 ml/kg of 1% sodium pentobarbital solution (Shanghai New Asiatic Pharmaceuticals Co., Ltd., Shanghai, China). Next, the biceps femoris in the hindlimb of the rats was located. A tight band with a diameter of approximately 2–3 mm or more could be felt with our fingers over the right medial femoral muscle. This specific area was marked as the therapeutic area for silver needle thermal therapy.
After disinfecting the rats, a silver needle (length: 10 cm, diameter: 0.6 mm) was pierced at the starting and end point, and the tension zone of the rat's right femoral muscle until the tip reached the surface of the femur. Silver needle thermal therapy was carried out to heat the needle and the temperature was set to 110°C and the heating time was 15 min. After the silver needle was removed, the puncture site was routinely sterilized with 75% alcohol and covered with an aseptic dressing. Afterward, the rats were returned to their respective cages for normal feeding.
Thermal hyperalgesia test for thermal pain threshold
Seven days after the MPS rats had undergone silver needle thermal therapy, the paw withdrawal latency was employed to detect the pain threshold of the rats. The Hargreaves Plantar test was performed as previously described using a standard apparatus (Ugo Basile, Italy). A thermal radiant stimulus was positioned underneath the targeted hind-paw and the latency of the paw withdrawal response was automatically measured.[16] The stimulation was automatically terminated at 40 s in refractory animals to avoid tissue injury. The paw withdrawal latency of each hind paw was determined and recorded. The procedure was performed thrice on each rat, with the rats allowed to rest for more than 15 min between each trial. The average value was calculated and regarded as the paw withdrawal latency.
Von Frey hair test for mechanical pain threshold
To test the stable existence of persistent pain, the rats were placed in a plexiglass lattice (26 × 20 × 14 cm3) individually and acclimatized for 20 min at the outset of each test. According to the up and down method,[17] we adopted von Frey filaments (Stoelting, Wood Dale, IL, USA) to assay the mechanical sensitivity two hours after the thermal pain threshold test was completed. Finally, we transformed the record into data and acquired the 50% paw withdrawal mechanical threshold.[18]
Histopathological examinations
After the von Frey hair test was finished, tissue specimens were obtained from the right medial femoral muscle, promptly placed in 4% paraformaldehyde (Solarbio, Beijing, China) to avoid destruction, and fixed at room temperature for one week. Next, the muscle specimens were dehydrated, removed, and embedded in paraffin. The paraffin blocks were then sectioned and dewaxed, and underwent hematoxylin and eosin (H&E) staining (Solarbio, Beijing, China). The morphology and arrangement of the muscle fibers were observed under an optical microscope.
Immunofluorescence staining
After the von Frey hair test was finished, spinal cord sections were deparaffinized, rehydrated in a series of graded ethanol, and then incubated in 0.3% H2O2 for 10 min. After antigen retrieval, the sections were permeabilized with 0.2% Triton X-100, and then incubated with 2% fetal bovine serum (FBS, Solarbio, Beijing, China) for one hour at room temperature. Next, the sections were incubated with the primary antibody, that is, rabbit antibodies to 5-HT3R (dilution ratio of 1:3000) (Abcam, UK) overnight at 4°C. After washing, the sections were incubated with the secondary antibody, that is, Fluorescein isothiocyanate (FITC)–conjugated Affini donkey anti-mouse IgG (H + L), Alexa Fluor 594–conjugated donkey anti-mouse (Abcam, UK) for two hours at room temperature. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with 4',6-diamidino-2-phenylindole (1:5000) for five minutes, mounted onto glass slides, and cover-slipped with an antifade mounting medium. Images were then acquired using a laser scanning confocal microscope.
Western blot assay
After the von Frey hair test was finished, spinal cord tissues were lysed in radio-immunoprecipitation assay lysis buffer supplemented with a protease inhibitor cocktail (Solarbio, Beijing, China). The protein concentration was subsequently determined using a bicinchoninic acid assay. Next, 20 μg of total protein lysate were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking in 5% skim milk, the membranes were probed with antibodies against 5-HT3R (dilution ratio of 1:500, Abcam, UK) overnight at 4°C and detected by incubating with specific secondary antibodies for one hour at room temperature. Bands were later visualized using enhanced chemiluminescence detection reagents and quantified with the ImageJ software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control.
Statistical analysis
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 21.0 software (IBM Corporation, Armonk, NY, USA). Measurement data were expressed as mean ± standard error (SE) of the mean. Mean difference was regarded significant at the 0.05 level. For comparison of data obeying normal distribution and homogeneity of variance among multiple groups, one-way analysis of variance (ANOVA) was performed. Comparison between two sample means was conducted with t-test after homogeneity test of variances.
ResultsSilver needle thermal therapy augmented the thermal pain threshold as well as mechanical pain threshold of MPS rats
To detect the thermal hyperalgesia and mechanical pain threshold of the rats, we examined the paw withdrawal latency of each hind paw and paw withdrawal mechanical threshold. As shown in [Figure 1], the thermal and mechanical pain threshold of MPS rats were both found to be significantly lower, indicating that the MPS models were successfully established. Compared with the MPS model group, the pain threshold of rats in the silver needle thermal therapy group was markedly increased, which suggested that silver needle thermal therapy can reduce thermal and mechanical hyperalgesia in the MPS rats.
Figure 1: The paw withdrawal latency and paw withdrawal mechanical threshold in each group. (a) The paw withdrawal latency of each hind-paw. (b) The paw withdrawal mechanical threshold. The paw withdrawal latency of each hind-paw as well as the paw withdrawal mechanical threshold of MPS rats was significantly decreased (P < 0.01). Compared with the MPS model (MDC) group, the paw withdrawal latency of each hind-paw and the paw withdrawal mechanical threshold of rats in the silver needle thermal therapy (SNT) group were significantly increased (P < 0.01). This showed that the pain threshold of rats in the MDC group was significantly decreased, while it was increased in the SNT groupSilver needle thermal therapy repaired the damaged muscle fibers at the trigger point of MPS rats
In order to investigate the pathomorphological changes in each group, H and E staining was adopted to observe the morphological changes of the right medial femoral muscle fibers in each group of rats. It was found that in the normal control group, the transverse section of the muscle appeared in either a uniform round or irregular structure, which was arranged closely and regularly. Meanwhile, the transverse sectional section of muscle tissue in the MPS model group exhibited atrophy, degeneration of muscle fibers, as well as different sizes of elliptical and round-shaped muscle fibers. In the silver needle thermal therapy group, a small number of muscle fibers that were slightly atrophied and denatured were observed, while the shape was similar to that of the normal group, which indicated that silver needle thermal therapy could repair damaged muscle fibers at the trigger point of MPS rats [Figure 2].
Figure 2: Pathomorphological changes. H and E staining was used to observe the morphological changes of the right medial femoral muscle fibers in each group of rats (a: Longitudinal section, b: Transverse section). In the normal control group, the transverse section of the muscle appeared in either a uniform round or irregular structure, which was arranged closely and regularly. The transverse sectional section of muscle tissues in the MDC group showed atrophy, degeneration of muscle fibers, as well as different sizes of elliptical and round-shaped muscle fibers. In the SNT group, a small number of muscle fibers that were slightly atrophied and denatured were found, and the shape was similar to that of the normal group, which showed that silver needle thermal therapy could repair damaged muscle fibers at the trigger point of MPS rats5-HT3R is down-regulated in the spinal cord of the silver needle thermal therapy rats compared with the MPS rats
Immunofluorescence and western blot analysis were further carried out to assess the expression patterns of 5-HT3R in the spinal cord. Compared with the NRC group, the expression levels of 5-HT3R were markedly elevated in the MDC and SNT groups. Meanwhile, 5-HT3R expression levels in the MDC group were significantly up-regulated compared with those in the SNT group [Figure 3] and [Figure 4].
Figure 3: Expression of 5-HT3R in the spinal cord by immunofluorescence. Compared with the NRC group, the expression of 5-HT3R in the spinal cord of the MDC and SNT groups showed a significant increase (P < 0.01). The expression of 5-HT3R in the spinal cord of the SNT group was significantly down-regulated compared with that in the MDC group (P < 0.01)Figure 4: Expression of 5-HT3R in the spinal cord by western blot. Compared with the NRC group, the expression of 5-HT3R in the spinal cord of the MDC and SNT groups showed a significant increase (P < 0.01). The expression of 5-HT3R in the spinal cord of the MDC group was significantly up-regulated compared with that in the SNT group (P < 0.01) DiscussionThe current study was conducted by establishing MPS rat models and treating the MPS rats with silver needle thermal therapy; simultaneously, a normal control group was set up. We subsequently detected the pain threshold of each group of rats, and the pathological changes of the muscle fibers at the trigger point. In addition to this, we also detected the expression patterns of 5-HT3R in the spinal cord of each group of rats.
MPS is well-recognized as a regional muscle pain disorder and featured as the leading cause of persistent regional pain.[3] Unfortunately, MPS is frequently overlooked as a clinical diagnosis because it is often accompanied by various signs and symptoms in addition to pain, coincidental pathological conditions, and behavioral and psychosocial problems.[19] Currently, management of MPS encompasses palliative care, splint therapy, muscle exercises, therapy to the trigger points, and behavioral therapy depending upon the complexity of the case. Although pain point injections and shock wave therapeutic strategies exhibit good immediate effects, the long-term effects remain unsatisfactory, especially for patients with chronic refractory MPS.[20],[21],[22],[23] Meanwhile, the last few years have also witnessed the adoption of acupuncture therapy for treating MPS patients. The short-term effect of acupuncture is fairly obvious, and even the long-term effects are more efficacious than other treatment methods. However, due to the long treatment duration and cycle, as the frequency of acupuncture increases, there is a surge of acupuncture tolerance, which can reduce its analgesic effects.[24]
Nevertheless, we have found in clinical practice that silver needle thermal therapy has a better curative effect, especially for patients with chronic refractory MPS while the long-term curative effects are also fairly significant. Inherently, silver needle thermal therapy has evolved from the Nine Needles of traditional Chinese medicine, such that it strictly follows the surgical anatomy of human soft tissue and the distribution of soft tissue tenderness points. Different from normal acupuncture practice, silver needle thermal therapy is performed with the help of precise silver needles and introduces the best temperature required in an effort to eliminate aseptic inflammation, relax muscle spasms, increase local blood supply, promote tissue repair and muscle cell regeneration, and consequently relieve soft tissue pain. Compared to stainless-steel acupuncture needles, silver needles have a thicker body and silver content reaching up to 80%. Moreover, the heat conduction effect of the silver needle tail after heating is approximately 23 times that of milli needles (stainless-steel needles), while the needle penetration depth of silver needles is far deeper than that of stainless-steel acupuncture needles. As a result, the use of silver needle thermal therapy allows the transfer of heat energy to deep diseased tissues to elicit a therapeutic effect.
In our study, the results of H and E staining demonstrated that the cross-section of the muscles of the rats in the normal group presented with a more uniform circular or irregularly shaped structure and the longitudinal section exhibited muscle fibers that were tightly-arranged and regular. Meanwhile, the cross-section of the muscle tissue of the model group exhibited muscle fiber atrophy and degeneration, and oval and round muscle fibers of various sizes, whereas the longitudinal section was observed to present with disordered muscle fiber arrangement. Moreover, we found that there was only a small amount of muscle fiber atrophy and degeneration on the cross-section of the SNT group, while the longitudinal section showed no obvious disorder in the arrangement of muscle fibers and the morphology was close to the normal group. Altogether, these findings indicated that silver needle thermal therapy could promote the repair of muscle fibers at the trigger point in MPS rats.
Furthermore, we also adopted the thermal hyperalgesia test and von Frey hair test to detect the thermal and mechanical pain threshold of each rat. The subsequent findings revealed that the thermal pain threshold and mechanical pain threshold of the MPS rats were both significantly lower than those in the normal rats, whereas the use of silver needle thermal therapy could significantly augment the thermal pain threshold and mechanical pain threshold of the MPS rats, suggesting that MPS rats present with central sensitization while silver needle thermal therapy can reduce this central sensitization of MPS rats.
The pathogenesis of MPS is highly-complex, with some studies confirming that continuous stimulation of MTrPs can lead to central sensitization; however, its specific mechanism remains elusive.[25] On a separate point, a number of researchers have turned to identify the role of 5-HT receptors in pain modulation in recent years. The hard work of our researcher peers has now revealed that the effects of 5-HT receptors to facilitate or inhibit pain transmission are closely-associated with the receptor subtypes and the sites of action.[6],[7] Moreover, previous studies have also revealed the involvement of 5-HT3R in the spinal cord in descending facilitation, which can further lead to central sensitization.[26] 5-HT3R confer a wide variety of functions, including pain modulation. In addition to this, 5-HT3 receptors were previously shown to contribute to any central plasticity that accompanies this injury state, and the hyperalgesia and allodynia that manifests after tissue injury involves different peripheral and/or central mechanisms.[27] In our study, we found that 5-HT3R were highly expressed in the spinal cord of the MPS rats, which is suggestive of the potential involvement of 5-HT3R in the process of central sensitization of MPS. Additionally, our findings further revealed that silver needle thermal therapy could significantly diminish the expression levels of 5-HT3R in the spinal cord of MPS rats. Collectively, these findings and data suggested that silver needle thermal therapy may exert an advantageous effect by reducing the expression of 5-HT3R in the spinal cord, thus reducing the downward pain facilitation effect to alleviate central sensitization. However, the specific mechanism still warrants further exploration.
ConclusionsAltogether, the findings obtained in our study demonstrate that MPS rats exhibit myofascial edema and disordered muscle fiber arrangement at the trigger point, in addition to central sensitization and up-regulated expression levels of 5-HT3R. More importantly, silver needle thermal therapy can repair damaged myofascial and muscle fibers, increase pain threshold, and reduce the expression of 5-HT3R in the spinal cord of MPS rats.
Acknowledgments
We would like to give our sincere appreciation to our colleagues for their technical assistance and valuable suggestions on this article.
Financial support and sponsorship
Adult Sprague–Dawley male rats (weighing 200-250 g; Guizhou Medical University Animal Center, Guiyang, China) were included for the current study. The procured rats were individually housed in a temperature-controlled room, with natural light and relative humidity of 40%–60%. Animal experiments were carried out according to the guidelines of laboratory animal care, and experimentation protocols were approved by the Institutional Animal Care and Use Committee of Guizhou Medical University. Extensive efforts were made to minimize the number and suffering of the experimental animals.
This work was supported by the Natural Science Foundation of China (Grants No: 82060811/H2902) and Guizhou Province Science and Technology Plan Project (Grants No: Qianke He Foundation -ZK(2021)General 508).
Conflicts of interest
There are no conflicts of interest.
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