| Objective To observe the effects of remote and proximal point selection on Substance P(SP),Calcitonin gene related peptide(Calcitonin gene related peptide),Myofascial pain syndrome(MPS)rat model.The effects of CGRP,macrophages and inflammatory factors on peripheral analgesia in myofascial pain syndrome were compared.MethodsTwenty-four healthy male SD rats(SPF grade,7 weeks of age,weight 200±20g)were randomly divided into blank group,model group,proximal point selection group(hereinafter referred to as proximal group)and Yanglingquan and Yinlingquan group(hereinafter referred to as distal group),with 6 rats in each group.The chronic MPS model in model group,proximal group and distal group was prepared by "blow combined with centrifugal exercise".The specific methods were as follows:On the first day of the experiment,the rats were given inhalation anesthesia induced by 4%isoflurane using an animal gas anesthesia machine,and then the isoflurane concentration was adjusted to 2%.The rats were hit once with a self-made percussion device on the medialis muscle of the right lower limb with a kinetic energy of 2.352 J,and then returned to the cage for normal feeding.On the second day of the experiment,the rats were continuously run downhill for 90 minutes at a speed of 16m/min on an electric treadmill at-16°,and were driven away by electric shocks and small sticks.The remaining 5 days of normal feeding,no intervention.The intervention period was once a week for 8 weeks.After that,normal feeding for 4 weeks,without any treatment,is the recovery period.After the modeling was completed,the rats in the proximal group were searched for the pain point,and the 0.30mm×25mm millimeter needle was used for oblique piercing and penetrating through the pain point nodules.After the local convulsive reaction was induced by lifting and twisting,the Korean electroacupuncture instrument was connected,and the density wave with a frequency of 2/100 Hz and a voltage of 2-4V was applied.The current intensity was appropriate for slight tremor in the right lower limb of the rats,and the treatment was carried out for 15 min,once a day,continuous treatment for 14 days.The rats in the distal group were needled at Yanglingquan point plus Yinlingquan point in the right lower extremity,the same as the proximal group;The rats in blank group and model group were grasped and fixed in the same way at the same time and place without any treatment.The mechanical pain threshold and musculoskeletal ultrasound imaging were measured before,after and after the intervention.After intervention,the 4 groups of rats were tested by electromyography.HE staining was used to observe the morphological changes of the medial femoris muscle of the right lower limb of rats.The positive expressions of SP,CGRP,M1-type macrophage marker CD68 and M2-type macrophage marker CD206 in muscle tissue were observed by immunofluorescence histochemistry.Enzyme-linked immunosorbent assay(ELISA)was used to detect Interleukin-1β(IL-1β)and Interleukin-6(interleukin-6)in serum.The content of IL-6,Tumor Necrosis Factor-α(TNF-α),Interleukin-10(IL-10),and Interleukin-8(IL-8).Results1.Vonfrey detects the mechanical foot retraction reflex thresholdBefore modeling,there was no significant difference in the threshold of mechanical foot retraction reflex among all groups.After modeling,the mechanical foot retraction reflex threshold of rats in all groups was significantly decreased compared with that in blank group(P < 0.05).After intervention,the mechanical foot retraction reflex threshold of rats in the distal group and the proximal group was significantly increased compared with the model group(P < 0.05),but the proximal group was more significant than the distal group.2.Muscle tissue morphology was detected by musculoskeletal ultrasonography in small animals Musculoskeletal ultrasonography showed that the muscle shape of each group was complete,without fracture or distortion,with clear outline and orderly arrangement of muscle fibers.Compared with the blank group,the muscle fibers in the model group were broken,twisted,deformed,disordered and the outline was not clear.After the intervention,the arrangement of muscle fibers in the distal group and the proximal group was relatively orderly,and the fracture,deformation and distortion were reduced,but there was no significant difference in the morphological comparison between the two groups.3.Muscle electrophysiology detector was used to detect muscle electrophysiology at MTr Ps Touch the impact position of the medial femur muscle and its nearby muscles to determine the location of the pain point.Insert two fine needle electrodes into the nodule of the pain point(the reference electrode is inserted into the right medial femur muscle),and open 1-2cm beside the two needles to record the electromyography signal in the resting state for 10 minutes.The results of electromyography showed that there was no spontaneous discharge in the blank group at rest.The potential of high frequency and high amplitude appeared in the model group,and the duration was longer.The frequency and amplitude of spontaneous discharge in the proximal group were significantly lower than those in the model group.The background potential of high frequency and low amplitude and the spike of high amplitude and low frequency appeared in the distal group.4.HE staining was used to detect the changes of muscle tissue morphology(1)Horizontal incision of medialis muscleBlank group: the shape of myocytes was regular polygon,the size was relatively uniform,the myofibril was tightly arranged,the nuclei were all located under the myomedium,the color was uniform,the arrangement was orderly,there was no accumulation of nuclei,no obvious inflammatory cell infiltration,and no blood cells.In the model group,the myocytes were irregular in shape,loosely arranged and irregular,the muscle space was irregular and widened,the structure was chaotic,the cells were obviously enlarged and rounded,the nuclei were inwardly shifted and the size was uneven,the color was deep,the nuclei were clustered in multiple places,there was obvious inflammatory cell infiltration,and a few blood cells were visible.In the proximal group,the shape of the muscle cells was restored to regular polygon,the size was relatively uniform,the muscle space was relatively tight,the structure was clear,the nucleus was located under the muscle membrane,there was no obvious inward migration or aggregation phenomenon,and the size and coloring of the nucleus were relatively uniform.In the distal group,the shape of the muscle cells was restored to regular polygon,the size was relatively uniform,and the muscle space was relatively tight,but the cell nucleus was looser than that in the proximal group,and the nucleus was located under the muscle membrane,without obvious inward movement,with slight nucleus aggregation phenomenon,and the nucleus was still deeply stained.(2)Coronal section of vastus medialis muscleIn the blank group,the muscle fibers were arranged neatly and continuously,the fiber thickness was relatively uniform,the nuclei were uniformly arranged in line between the muscle fibers,and no inflammatory cell infiltration was observed.Model group: the arrangement of muscle fibers was disordered,twisted and broken,the fiber thickness varied,and there was nucleus aggregation.In the proximal group,the muscle fibers were arranged relatively neatly,there was no obvious muscle fiber breakage,the fiber thickness was relatively uniform,there was no inflammatory cell infiltration,but there was still a small amount of cell aggregation.In the distal group,the muscle fibers were arranged relatively neatly,a few muscle fibers were broken,the fiber thickness was relatively uniform,there was no inflammatory cell infiltration,some nuclei were disordered,and there was a small amount of cell aggregation.5.The expression results of SP,CGRP,CD68 and CD206 in rat medial femoral muscle were detected by immunohistochemistryCompared with the blank group,the levels of SP,CGRP,CD68 and CD206 in the local muscle tissue of the model group were significantly increased(P < 0.01);compared with the model group,the level of SP in the proximal group was decreased(P < 0.05),the levels of CGRP and CD68 were significantly decreased(P < 0.01),and the level of CD206 was significantly increased(P < 0.01).There was no significant difference in SP and CGRP in the distal group,CD68 level was significantly decreased(P < 0.01),CD206 level was significantly increased(P < 0.01).6.Serum levels of IL-6,IL-8,IL-10,IL-1β and TNF-α were detected by ELISACompared with the blank group,the levels of TNF-α and IL-8 in the model group were significantly increased(P < 0.01),while the levels of IL-6,IL-10 and IL-1β in the model group were not statistically significant.Compared with the model group,the levels of IL-8 in the proximal group and the distal group were significantly decreased(P < 0.01),and the differences in TNF-α were not statistically significant.ConclusionsProximal point selection can improve the pain threshold of rats and reduce SP,CGRP and other paincausing substances.The mechanism of peripheral analgesia may be through reducing the release of local IL-8,thereby reducing the levels of SP and CGRP,and promoting the polarization of macrophages.The peripheral analgesia effect of remote point selection is not obvious,and the mechanism is still unclear. |
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