| Background:Myofascial pain syndrome(MPS)is a symptom of the sensory nerve,motor nerve and autonomic nervous system caused by myofascial pain trigger points.This is clinically called myofascial pain syndrome.MPS is a common type of chronic pain identified by myofascial trigger points(MTrPs).MTrPs are considered hypersensitive areas over a taut band of skeletal muscle.The ethology and mechanism of MTrPs remains under study.Sustained low-intensity muscle contractions,such as posture naintenance,can lead to luscle damage and Ca2+ homeostasis disruption,which create MTrPs.However,the mechanism on how these trigger points cause pain remains unclear.Myofascial pain is a complex form of neuromuscular dysfunction that consists of motor and sensory abnormalities,which involve both the peripheral and central nervous systems.The tissue injury-induced sensitization of peripheral nociceptors is characterized by an exaggerated pain response to noxious stimuli,which is called,peripheral sensitization.Furthermore,peripheral pain information activates sensory nerve endings that transmit acute nociceptive information to the dorsal horn of the spinal cord,which in turn induces central sensitization.This may be the primary mechanism for converting acute pain into chronic pain.Many studies support the argument that the activation of the receptor tyrosine kinase(RTK)system and its downstream signaling pathway are involved in the development of peripheral sensitization.Fibroblast growth factor receptor(FGFR1)belongs to the receptor tyrosine kinase(RTK)family,FGFR1 is constitutively expressed in the dorsal root ganglia and peripheral nerve,and is upregulated following injury.Fibroblast growth factor 2(FGF2,bFGF)activates the downstream signaling by binding to FGFR1.FGF2 concentrations have been widely reported to increase during wound healing.Elevated FGF2 levels have also been observed in diseases associated with pain sensitization,such as adjuvant-induced rat arthritis.As a protective mechanism for the body,FGF2 and FGFR1 cause pain sensitivity while repairing tissue damage,and FGF2 injections in the feet of rats reduced the mechanical pain threshold.A large body of evidence supports the notion that inhibiting FGFR1 relieves pain.The expression of FGFR1 in the dorsal root ganglia is also correlated to peripheral pain,and gabapentin relieves arthritis pain by reducing the expression of FGFR1 in the dorsal root ganglia of rats.FGFR1 in the sciatic nerve of mice is associated with thermal pain sensitivity,and the selective loss of FGFR1 signaling causes a decrease in sensitivity to heat pain.This study shows that the expression of FGFR1 on the sciatic nerve may be correlated to peripheral sensitization.The PI3K/AKT pathway is the downstream signaling pathway of FGFR1.The PI3K/AKT pathway plays an important role as a downstream pathway for many receptors in pain sensitization.Phosphoinositide 3-kinase(PI3K)is indispensable in the development and maintenance of chronic pain.As a downstream signaling molecule of PI3K,phosphorylated AKT(p-AKT),can be used as an important indicator of PI3K activation.Therefore,it was hypothesized that p-FGFRl may mediate myofascial pain though the PI3K/AKT pathway in rats.A human study and some animal experiments were performed to test this hypothesis.Part ?:Expression of P-FGFR1 at the trigger point of human trapeziusObjective:This study aims to detect the expression of p-FGFR1 in the control group and myofascial pain group.Materials and Methods:1.PatientsPatients diagnosed with myofascial pain by the Department of Orthopaedics of Qilu Hospital and controls without pain-related symptoms were recruited.The diagnosis of MPS was performed by the same operator to avoid the differences caused by different operators,and this was determined by a physician with more than 20 years of clinical experience by applying the published diagnostic criteria,including an inquiry into the history of the disease and a physical examination.The inclusion criteria for patients in the present study were,as follows:men or women between 18-65 years old,who were diagnosed with primary myofascial pain in the upper trapezius muscle.The exclusion criteria were recent trauma,pregnancy,arthritis,diabetes,or other diseases.Based on their history and physical findings in the upper trapezius muscle,11 participants were recruited into the MTrPs group(M group),and seven participants were recruited into the control group(C group).The potential myofascial trigger point was excluded.A review of the medical history of these study participants confirmed that no participant was treated with radiation or chemotherapy,or with cervical radiculopathy.2.Muscle biopsyThe invasive operation has been approved by Research Ethic Committee of Qilu Hospital(KYLL-2014-027).All participants provided a signed voluntary informed consent in strict accordance with clinical practice.The muscle samples were taken from the taut band or nodule of the upper trapezius muscle,which contained the MTrPs,according to Bergstrom's standard procedure.The site was anesthetized by injecting 1.5 ml of 0.5%lidocaine.After local anaesthesia,and when the acupuncture made the subject feel the same severe pain as the previous pain symptoms,and visually observation of the local muscle contraction response was performed,the tip continued to pierce 3 mm(the distance from the tip to the tissue groove was 3 mm),allowing the tissue to remian inside the groove.The muscle sample was immediately biopsied using a Bergstrom biopsy needle.Then,this was frozen in liquid nitrogen and stored at-80?,3.RTK phosphorylation antibody microarray analysisThe p-FGFR1 antibody arrays used the RayBio Human Phosphorylation Array Kit(catalogue no.AAH-PRTK-G1),which was purchased from RayBiotech Inc.(Guangzhou,China).The performance of the p-RTK array was performed according to manufacturer's instructions.Briefly,100 ?l of 1× blocking buffer was added to each well,and incubated for 30 minutes at room temperature with gentle shaking,in order to block the slide.Then,the buffer that blocked each well was decanted(in order to ensure that all buffers are removed).Afterwards,100 ?l of each sample was added to the wells.Next,the samples were incubated at 4? overnight.Then,the sample in each well was decanted,and the wells were gently washed for three times with 100 ?l of 1× Wash Buffer I at room temperature.Afterwards,100 ?l of 1×biotin-conjugated anti-phosphotyrosine was added to each of the corresponding wells,and incubated with gentle shaking at room temperature for two hours.After washing for three times,100 ?l of 1× fluorescent dye-bound streptavidin was added to each subarray,washed for three times,and completely dried.Then,the signals were imaged using a laser scanner.4.Statistical analysisThe data are expressed as the meanąstandard deviation(SD).Then,the data was analyzed using the SPSS 18.0 statistical software.A statistical analysis between two groups was performed using Student's t-test.Results that were described as significant were based on a criteria of P<0.05.Results:In analyzing the phosphorylated protein expressed in the muscle tissue in the MTrPs group,the p-FGFR1 increased,when compared to the control group(P<0.05).Conclusion:This result indicates that p-FGFR1 is involved in human myofascial pain.In order to explore the mechanism of the p-FGFR1 involved in myofascial pain,it is necessary to establish a rat myofascial model for further research.Part ?:Establishment of the rat gastrocnemius myofascial pain modelObjective:This study aims to establish a model of gastrocnemius myofascial pain in rats,and explore the mechanism of myofascial pain in rats.Materials and Methods:1.Experimental animalsMale Sprague-Dawley rats(six weeks old)were used for the present study.These rats(weighing 200-250 g)were housed(three rats per cage)with a controlled relative humidity(20-30%)in a 12-hour light-dark cycle at room temperature(24?).Food and water were freely accessible.2.Establishment of the myofascial pain modelIn the present study,the investigators used the same animal model,as reported by Huang et al.The rat model of active MTrPs was established by a blunt strike on the left gastrocnemius muscle(GM)and eccentric-exercises for eight weeks,along with four weeks of recovery.The left GM of rats in all the groups was marked and struck by a hand-made stick device dropped from a height of 20 cm,with a kinetic energy of 2.352 J,on the first day of each week.Then,these rats were run on a treadmill(Sans,Nanjing,China)for 1.5 hours at a-16° downward angle and a speed of 16 m/min every second day of each week.The general health of these rats during the procedures,such as body weight,limb condition and movement,was monitored weekly.In order to identify the active MTrPs,a contracture in a taut band was palpated and marked.If a local convulsion response(LTR)was caused by acupuncture,the marked nodules were considered possible active MTrPs.3.Measurement of rat tenderness thresholdA tenderness meter equipped with a round head probe was used to measure the mechanical tenderness threshold,and the hind limb withdrawal threshold in response to the mechanical stimulation of the left hind leg was the tenderness threshold.Before the start of the study,these rats were acclimatized in a laboratory environment for seven days,during which the rat tenderness threshold was measured.When measuring the tenderness threshold,the rat was restrained in a cylinder,and the pressure pain meter probe was applied to the left gastrocnemius muscle.In the Randall-Selitto test,the pressure would automatically increase until the rat shrunk its feet or makes a call.Seven measurements were taken,and the average value after removing the minimum and maximum values was used as the tenderness threshold.The rat's basic pain threshold,the pain threshold at 1-6 days after the first hit,the pain threshold one one day before the weekly hit,and the pain threshold during the recovery period were messured.4.Hematoxylin-Eosin(H&E)stainingThe muscle specimens of rats in the my ofascial pain group and control group were fixed in formalin.Subsequently,a series of routine procedures were used to process the specimen,including dehydration,paraffin embedding,and sectioning.A section with a thickness of 4 ?m was attached to a glass slide,and deparaffinized and stained with H&E.Then,the sections were transparently dehydrated in gradient ethanol(70-100%)and xylene,and covered with a cover glass.Afterwards,the slices were observed using an optical microscope equipped with a digital camera.Results:1.Mechanical hyperalgesia in MTrPsThe mechanical withdrawal thresholds measured using a Randall-Selitto apparatus in rats with MTrPs(M group)(n=10)significantly decreased,when compared with those for control rats(n=8),from week 3 through week 12.The pain thresholds continued to decline from week 3 to week 8,and maintained a low level through week 12.The control animals exhibited no changes,when compared to the baseline threshold.2.Muscle histologyBased on the microscopic analysis of the H&E staining,the muscle fibres in the control group were uniform in size,polygonal,and regular in arrangement in the cross-sectional space.Large round muscle cells(contracture knots)could be observed in the cross-sections of the MTrPs.On the profile,the muscle fibres were regular and uniform in thickness.The continuous expansion of pyramidal muscle fibres was observed in the longitudinal sections.The muscle fibre space of the cross-sections and longitudinal sections increased.In the MTrPs,internal nuclei cells were observed.Conclusion:The changes in pain threshold,the changes in muscle morphology,the appearance of a nodular tension zone,and local twitch response(LTR)proves that the rat myofascial pain model was successfully established.Part ?:Expression and mechanism of P-FGFR1 at the trigger point of gastrocnemius muscles in ratsObjective:The expression and mechanism of FGF2/p-FGFR1 at the trigger point of gastrocnemius muscles in rats were detected.Materials and Methods:1.Animal groupingAfter establishing the myofascial pain model of rats,these rats were randomly divided into four groups:normal+DMSO,MTrPs+DMSO,MTrPs+FGFR1 inhibitor PD173074(Selleck,2.5 mg/ml),and MTrPs+PI3K inhibition LY294002(MCE,1 mg/ml)groups.2.Pharmacology.The effects of the administration of PD 173074 and LY294002 on the left hind limb withdrawal threshold in response to mechanical stimulus were explored in rats with MTrPs on week 13.PD 173074 and LY294002 were intramuscularly administered to rats in the MTrPs group(30 ?l × three points per rat).The mechanical withdrawal threshold was measured using the Randal-Selitto apparatus at 0.5,1,2,4,6,8 and 24 hours after intramuscular administration.The effects of the vehicle(1%DMSO)were also examined by administering the control at the same volume as the inhibitors.After the injection of PD 173074 of 2.5 mg/ml,these rats were anaesthetized with chloral hydrate(i.p.),underwent sternotomy,and were intracardially perfused with physiological saline.Then,the left gastrocnemius muscles near the injection site were quickly extracted.One part of the muscles was post-fixed in 4%paraformaldehyde for 24 hours,and subsequently used for the H&E and immunohistochemistry,while the other part of the muscles was stored in liquid nitrogen for western blot analysis.The L4-L5 dorsal root ganglia(DRG)and L4-L5 spinal cords of rats were quickly extracted for the immunohistochemistry.3.Statistical analysisThe data was expressed as meanąstandard deviation(SD).The SPSS 18.0 statistical software was used to analyze the data.One-way analysis of variance was used for comparisons among the three groups,and t-test was used for the statistical analysis between two groups.P<0.05 was considered statistically significant.Results:1.The expression level of FGF2 in the muscle cell gap in the MTrPs group measured by immunohistochemistry was higher,when compared to that in the control group.In addition,as determined by the western blot analysis,the protein expression of FGF2 in MTrP was upregulated,when compared to the control group.These results indicate that FGF2 is released from damaged muscle cells in MTrPs,which may cause pain sensitivity.2.The immunohistochemistry experiment of p-FGFRl was performed at one hour after the injection of 1%DMSO.The peripheral nerve fibers were labeled with PGP9.5.The immunohistochemistry revealed that the expression of p-FGFRl in the nerves increased in the MTrPs group,when compared to the control group.The expression of p-FGFRl in the L4-5 dorsal root ganglion(DRG)was higher in the MTrPs group,when compared to the control group.In addition,the Fos expression in the dorsal horn of the spinal cord increased in the MTrPs group.The Fos expression in the dorsal horn of the spinal cord was used as a marker of muscle pain.P-FGFR1 was expressed in DRG neurons,and the expression of p-FGFR1 in DRG neurons in the MTrPs group increased,suggesting that the FGFR1 receptor in DRG is activated by the FGF2 released from the muscle.These results indicate that p-FGFR1 in peripheral nerves and DRG neurons is involved in the maintenance of myofascial pain.3.The intramuscular injection(i.m.)of PD173074(2.5 mg/ml)increased the mechanical pain threshold in the MTrPs group at 0.5,1,2 and 4 hours after injection,and the analgesic effect peaked at one hour.LY294002(1mg/ml)increased the mechanical pain threshold in the MTrPs group at 0.5,1,2 and 4 hours after injection,and the analgesic effect peaked at 0.5 hours.4.Compared to the control group,the protein expression of p-FGFR1,PI3K-p110?and p-AKT was significantly up-regulated in the MTrPs group.According to the results of these present behavioral experiments,since the analgesic effect of PD 173074(2.5 mg/ml)peaked at one hour,the investigators chose a time point of one hour for the western blot analysis.Compared to the MTrPs group at one hour after injection,PD173074(2.5 mg/ml)suppressed both the expression of p-FGFRl,and the expression of PI3K-p110? and p-AKT.Conclusion:1.FGF2 is released from damaged muscle cells in MTrPs,activating its receptor FGFR1.2.The nerve fibers near the trigger point and p-FGFRl in DRG neurons are involved in the maintenance of myofascial pain.3.The FGFR1 receptor on nerve fibers near the trigger point is activated,and regulates myofascial pain in rats through the PI3K/AKT pathway. |
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