Identification Of The Pathogenesis Of Chronic Myofascial Trigger Points Based On Muscle Spindles And Proteomics

Objective:In recent years,with the accelerated pace of the modern life,the development of sports and the coming of aging society,chronic pain caused by sports injury is gradually becoming one of the major problems affecting the style of life and production of urban residents and sports career of individual athletes.According to many years of clinical studies,most intractable and nonorganic-lesions pain are derived from skeletal muscle systems.The clinicians of Europe and the United States call them myofascial trigger points(MTrPs)pain.The pathogenesis of the disease has been a research focus in the field of pain,but it is not clear yet.Hubbard and Berkoff adopted unipolar needle electromyography to record high peak potential of MTrPs,and they thought the discharge activity probably came from abnormal muscle spindles.Also,in early we found that several abnormal peak inverted spontaneous potentials(PISP)can be observed in rest,which was markedly different from endplate potentials in normal skeletal muscle.So we speculated that the unusual potential may stem from the discharge of kernel chain fiber and nuclear bag in muscle spindles,the muscle spindle discharge approach.This study would explore the source of PISP in MTrPs to perfect lack of the predecessors from the perspective of H reflex,Ramp-and-hold stretch,and drug intervention.In addition,Simons puts forward the hypothesis that the contraction of myofascial trigger points could be located in the motor endplates,so it is very valuable to explore the antigenicity of the existence of nodular contracture.Thus,this study also would observe the morphologic changes of nodular contracture proposed by the isolated culture of MTrPs muscle cells and try to screen specific proteins associated with chronic MTrPs disease by iTRAQ proteomics,in order to provide a new direction for the precise treatment of MTrPs cells in immunobiology in the future.Methods:132 SPF male SD rats with the weight of 220g-250g were randomly divided into two groups(MTrPs group:82 rats,normal group:50 rats).Then the model rats were randomly divided into 11 groups,of which the first 10 groups with 8 rats in each group were used for H-reflex(Group A1),ramp-and-hold stretch(Group A2),succinylcholine injection(Group A3),eperisone hydrochloride injection(Group A4),saline injection(Group A5),blank drug intervention(Group A6),the observation of muscle spindles' morphology(Group C1),the detection of protein level of intrafusal NT-3 and TrkC(Group C2),the detection of gene level of intratusal NT-3 mRNA and TrkC mRNA(Group C3)and MTrSs cells culture(Group E1),and 2 rats from the last group were used for proteomics research(E2 group).The control group rats were randomly divided into 7 groups,of which the first 6 groups with 8 rats in each group were used for H reflex(Group B1),ramp-and-hold stretch(Group B2),the observation of muscle spindles' morphology(Group D1),the detection of protein level of intrafusal NT-3 and TrkC(Group D2),the detection of gene level of intratusal NT-3 mRNA and TrkC mRNA(Group D3)and normal muscle cells culture(Group F1 group),and 2 rats from the last group were used for proteomics research(Group F2).Rats model of chronic MTrPs was established by striking on fascia muscles and eccentric exercise,once per week,for continuous 8 weeks.Then those rats were reared normally and took rest for 4 weeks.At the end of the 12th week,those rats were examined for locating MTrPs with palpable taut band,the local twitch response and spontaneous electrical activity.On the basis of the successful modeling,the tibial nerves of two groups of rats were exposed and bipolar silver electrodes were used to stimulate the tibial nerves.Meanwhile,bipolar needle electrodes were used to record the EMG changes at the MTrPs and non-MTrPs,collecting the threshold to evoke H-reflex,the latency of M wave and H wave,Mmax,Hmax,and Hmax/Mmax.Secondly,based on the observation of PISP in MTrPs rats in Group A2,muscle spindles of gastrocnemius at the MTrPs and non-MTrPs were stimulated repeatly by the ramp-and-hold stretch,to observe the change of frequency and amplitude of PISP potential before-and after-stretch and on ramp phase and hold phase,and compared with the control group in Group B2.In addition,based on the observation of PISP in MTrPs rats in Group A3,A4 and A5,MTrPs was injected into by succinylcholine,eperisone and 0.9%saline to observe the change of frequency and amplitude of the abnormal PISP potential,and compared with blank intervention group.Besides,MTrPs muscles in Group C1 and non-MTrPs muscles in Group D1 were extracted to conduct the hematoxylin and eosin stain to observe the morphological changes of muscle spindle around MTrPs cells and non-MTrPs cells,respectively.The MTrPs muscles of gastrocnemius and non-MTrPs muscles of soleus,tibialis anterior and metatarsal muscles in Group C2 and C3 groups were extracted to detect the expression of protein and gene of NT-3 and TrkC by using Western blot and from polymerase chain reaction(PCR)tests,respectively,and compared with those in the conventional control group.MTrPs and non-MTrPs muscles in Group D1 and D2 were extracted to conduct the cell culture by using the method of the isolation of single muscle fiber.Subsequently,the possible MTrPs contracture nodules and normal muscle cells were intervened by different concentration of acetylcholinesterase and acetylcholine to observe the morphological changes from different muscle cells,respectively.Finally,iTRAQ proteomics technology was used to screen the differential proteins between MTrPs and non-MTrPs,and the correlation analysis of protein function and bioinformatics analysis(including GO function annotation,of KEGG pathway annotation,and so on)were performed.The statistical analysis was analyzed by the PRISM 5.01 software,and the results were presented as mean values and standard deviation(SD)for parametric data and median and interquartile range(IQR)for non-parametric data.Results:(1)The threshold(0.35±0.04mA)of the H-rcflex and latency(1.24±0.18ms)of the M wave recorded at MTrPs were lower than those at non-MTrPs(P>0.05).Compared with non-MTrPs,at MTrPs we could record the lower Mmax(4.28±1.27mV),higher Hmax(median[interquartile range]:0.95[0.80,1.08]mV)and Hmax/Mmax(median[interquartile range]:0.21[0.16,0.40]),and shorter H wave latency(4.60±0.89ms)(P<0.05).Ramp-and-hold stretch showed that the depolarization time of abnormal PISP was 0.4 to 0.9ms and the amplitude was 80?140?V.However,the abnormal PISP was not observed in ramp-and-hold stretch in control rats.The frequency of abnormal PISP in the ramp phase,hold phase and the 1st second after hold phase was significantly higher than that before ramp-and-hold stretch.After succinylcholine was injected into MTrPs,the amplitude and frequency of PISP first appeared a transient decrease,then increased rapidly,and reached the peak in 51-70s after injection.However,the amplitude and frequency of spontaneous endplate potentials decreased gradually with the extension of time,until it disappeared totally.After eperisone was injected into MTrPs,the amplitude and frequency of PISP decreased rapidly,and the wave frequency and amplitude of the endplate potentials were also significantly reduced,until they disappeared totally.In the 0.9%saline group,there was no significant difference in the amplitude and wave frequency of PISP in the whole observation period except for the transient increase of the amplitude and wave frequency immediately and 91-100s after injection.(2)the spindle capsules in MTrPs group were polygonal and some showed dimple phenomena,while the spindle capsules in the control group were round or oval and no dimple phenomena of capsules was observed.In addition,MTrPs cells with large round or ellipse shapes were much close to the neighboring muscle spindles.Results of Western Blot experiments showed that compared with non-MTrPs rats,the contents of NT-3 protein in the gastrocnemius muscle of MTrPs rats decreased significanty(P<0.05).Compared with non-MTrPs rats,the contents of NT-3 receptor TrkC protein in the medial gastrocnemius,toe muscles and tibialis anterior muscle of MTrPs rats decreased significantly(P<0.05).PCR results showed that compared with non-MTrPs rats,the contents of NT-3 mRNA in the medial and lateral gastrocnemius,toe muscles,tibialis anterior and soleus muscles of MTrPs rats decreased significantly(P<0.05).In addition,compared with non-MTrPs rats,the contents of TrkC mRNA in the medial and lateral gastrocnemius and toe muscles of MTrPs rats were also decreased significantly(P<0.05).(3)After the MTrPs cells culture,there still existed some large contracture nodules in muscle fibres,and some the contracture nodules are distorted.There was no obvious morphological change of the contracture nodules at the MTrPs cells after the treatment with different concentrations of acetylcholinesterase.There was also no significant morphological change in normal skeletal muscle after the treatment with different concentrations of acetylcholine in normal skeletal muscle cells.Compared with non-MTrPs,there were 50 differentially expressed proteins in MTrPs,in which the number of up-regulate and down-regulate expression and down was all 25.Conclusions:(1)Based on the H reflex pathway and ramp-and-hold stretch and drugs intervention of muscle spindles,it was found that sensitized spinal cord center and the excitability of Iaafferent nerves may be involved in the formation of MTrPs.muscle spindle sensitivity is increased.The PISP in the spontaneous potentials recorded at MTrPs was closely related to muscle spindles,so there was a close relationship between MTrPs and muscle spindles.(2)The formation of chronic MTrPs in the morphology could seriously affect the structure and function of muscle spindles and induced significant down-regulate expression of proteins and genes of NT-3 and TrkC at MTrPs and some related muscles.(3)MTrPs cells could be cultured to maintain the contracture in vitro,but it was not yet determined whether the recovery of the contracture nodules was related to acetylcholine.In addition,the differential proteins between MTrPs and non-MTrPs were mainly related to tumor diseases,energy metabolism disorders,the synthesis and binding of muscle cells proteins,muscle contraction,synapse,fibrinogen complex and rheumatoid arthritis.