| Objective:Fracture pain is one of the main symptoms of trauma,and also the first complaint when patients seek medical care.Especially for patients with limb fractures,the local inflammatory reaction caused by tissue damage is an essential part for debris cleaning and bone healing,but also would increase pain.Immobilization and reduction can help accelerate bone healing but cannot effectively prevent the formation of pain.Many patients would suffer from chronic pain even after fracture was healed.Except for anti-inflammatory and opioid drugs,there are no effective targeted treatment.Better control of pain can not only relieve the patient,but also help them do more functional exercise which promote the injury healing,so a good management of pain for patients after fracture is particularly important.In this project,a mouse fracture model was used to mimic the construction of a complex regional pain syndrome(CRPS)model,and a new specific microglia marker,Transmembrane protein 119(Tmem119)was evaluated in the development of chronic pain.The characteristics of activated microglia cells are analyzed in multiple time points in each sex,and the therapeutic effect was also compared after the antagonist treatment.Methods:We established the mouse CRPS model with closed distal fracture of the right tibia combined with cast fixation,and performed postoperative measurement of paw skin temperature,hind paw thickness,unweighting and von Frey test to evaluate the typical CRPS symptoms.In addition,we also used a recently described operant assay to demonstrate voluntary non-reflexive mechanical conflict avoidance(MCA)test to detect animal’s voluntary behavior in a more comprehensive and objective manner.The material was collected at multiple time points,including one day,one week,three week,and seven weeks after surgery.Then immunohistochemical staining and flow cytometry were used to qualitatively and quantitatively analyze the activated microglia in spinal dorsal horns with the expression of CD11b and Tmem119.In addition,Lipopolysaccharide rhodobacter sphaeroides(LPS-RS),an activation inhibitor of microglia targeting on TLR4,was injected intrathecally in both sexes,then the von Frey test were measured multiple times to check the drug effect.Results:After cast removal,CRPS mice showed significant signs of disease,such as increased skin temperature,edema of distal limbs,decreased bearing capacity and persistent pain of the ipsilateral side.The mean value of hind paw temperature is about 23.4±0.6℃,the average temperature of the ipsilateral hind paw in male mice is 1.5±0.2℃ higher than normal contralateral side,and 1.6±0.2℃ higher in female,there was no statistical difference between each sex(P>0.05).The average hind paw thickness before operation was 1.4±0.8 mm,and the difference between the ipsilateral and contralateral hind paw thicknesses of male and female mice was 0.36±0.08 mm and 0.45±0.02 mm,respectively.There was no statistical difference between each sex(P>0.05).In terms of hindlimb unweighting test,the post-operative weight-bearing values of male and female mice decreased to about 56±4.3%of the preoperative standard line,and no statistical difference between each sex as well(P>0.05).All affected hind paws showed hyperalgesia and allodynia.MWT decreased significantly at 3-9 weeks(P<0.001)and gradually improved after 9 weeks but didn’t return to preoperative level until 20 weeks later.There was no statistical difference between each sex(P>0.05).In MCA test,CRPS mice took longer to pass the test at 3 weeks post-operatively.Before the injury,the escape latency of male and female mice at the probe height of 5 mm was 15.8±1.7 s and 24.8±6.7 s respectively.After 3 weeks of modeling,the results of male and female mice were both more than 1 minute,which was significantly different from the baseline(P<0.01).At 2 mm,the escape latency of males was significantly increased(P<0.01),but not in females(P>0.05).It was confirmed that the chronic pain of CRPS mouse was slightly different in different sex.IHC results showed that microglial cells in the lumbar spinal cord dorsal horn were fully activated after CRPS modelling,with amoebic changes,and largely increased expression level of CD11b and Tmem119.The semi-quantitative method was used to calculate the percentage of the area of positive cells in dorsal horn region.The area of CD11b+cells in male mice reached a peak at 3 weeks after operation(P<0.001)and remained high to week 7.The expression of Tmem119 increased significantly at day 1(P<0.05)but decreased to a normal level after one week(P>0.05).However,female mice showed a delayed fashion in terms of glia acti vation.CD11b began to increase after 3 weeks,and peaked at 7 weeks,and there was no significant change in Tmem119.Furthermore,activated microglia were divided using flow cytometric according to the size,granularity and specific markers,and two subpopulations were obtained and quantified with differences between each sex.In male mice,the Tmemhi+cell increased significantly(P<0.001)while the Tmemmid+cell decreased significantly(P<0.001)after one day.After one week,the proportion of each cell return to preoperative level.However,in the early period of female mice,there was no significant change(P>0.05).After LPS-RS injection,male mice had a significant increase in MWT after one hour(P<0.05)and the relief lasted for 4 hours(P<0.001),then it dropped back to baseline level(P>0.05).Whereas the female mice showed no effect on this drug(P>0.05).Conclusions:By using a mouse model with cast fixation after distal tibial closed fracture,typical signs of CRPS were successfully and effectively mimicked.Mouse developed hyperalgesia in the affected limb with peripheral inflammation symptoms.With the rapid improvement of peripheral inflammation in the acute stage,the mice still showed persistent pain.With the activation of microglia in spinal cord,two types of cells were observed with different expression level of Tmeml 19,and the proportion of subtype cells varies with time and sex.The TLR4 competitive antagonist can only effectively relieve the pain in male.Therefore,the findings suggest that the microglial response to injury is not"one size fits all" but rather may vary depending on individual cell phenotype,timing after injury and sex of the subject,which could meaningfully guide the future studies of microglia modulation. |
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