complex regional pain syndrome type 1 ( crps1 ) is a disease of an extremity that usually 
 occurs as a complication after surgery or trauma , although spontaneous 
 occurrence is also described . three mechanisms are thought to be involved : afferent mechanisms ( e.g. , neurogenic 
 inflammation ) [ 24 ] , efferent mechanisms ( e.g. , 
 autonomic disturbances ) , and central nervous system 
 mechanisms ( e.g. , cerebral plasticity ) . 
 based on our review of the literature regarding the crps pathophysiology , we 
 hypothesized that following a trauma or surgery , the normal sterile 
 inflammatory response runs out of control , and is perhaps initiated by a 
 genetic and/or acquired immunologic disorder . neuroimmune activation of cells in the peripheral nervous system , which 
 is part of the afferent mechanism , apparently results in central sensitization 
 and exacerbation of pain . neuropeptides , cytokines , 
 and other mediators are released during the inflammation [ 9 , 10 ] and cause the prominent 
 signs and symptoms , which resemble inflammation ; these include increased skin 
 temperature , edema , pain , loss of function , and redness [ 7 , 11 ] . levels of tnf- and il-6 were previously shown to be elevated in fluid of artificially 
 induced skin blisters from the crps1 side in the initial stage of the disease [ 9 , 12 , 13 ] . we hypothesized that in most patients local inflammation 
 would only be present during the first year of the disease , since the clinical 
 signs and symptoms of crps1 are expected to diminish over time in most 
 patients . therefore , we predicted that the 
 formation of proinflammatory mediators ( such as il-6 and tnf- ) should 
 decline during the course of the disease . in an earlier study , we showed that 
 although the levels of tnf- and il-6 declined in the intermediate stage 
 of the disease , they were still significantly elevated in the crps1 extremity . 
 in the present study , we examine whether this decline in cytokine levels 
 continues during the course of the disease and whether this decline is 
 correlated with a possible improvement in disease activity as measured by 
 registration of pain , and by the measurement of differences in temperature , 
 volume , and mobility between the crps1 and contralateral extremities . the protocol was approved by the medical ethics committee of the erasmus mc 
 rotterdam ( mec no . guidelines according to the declaration 
 of helsinki ( amended version of 2002 ) and good clinical practice ( ich / gcp 
 version 1996 ) were followed . data collection and calculations were performed 
 according to guidelines for registration of personal data . sixty - six patients with crps1 in one extremity for 7 months 
 after the initial event participated in several studies conducted between april 
 2001 and february 2004 ( t0 ) to investigate the pathophysiology of crps1 or the 
 effects of specific treatments for crps1 [ 9 , 12 , 14 ] . at the time of the first follow - up study in 
 2004 , 25 patients with crps1 with a median disease duration of 30 months after 
 the initial event agreed to participate ( t1 ) . in 2007 , these 25 patients were 
 again asked to participate . one patient had died , one patient appeared to have 
 crps type 2 , and 11 patients chose not to participate again . in the end , for this study , we used the results obtained at baseline ( t0 ) , first 
 follow - up measurement ( t1 ) , and second follow - up measurement ( t2 ) from only 
 these 12 patients . all 12 patients fulfilled the crps1 criteria by bruehl et 
 al .   at the first measurement ( t0 ) , performed shortly after the 
 initial event ( median 4 months ) which resulted in the development of the 
 disease . the intensity of pain was assessed by using a visual 
 analoge scale ( vas ) recorded in 0100 millimeters . the mcgill pain questionnaire , 
 dutch language version ( mpq - dlv ) , was measured by counting the total number of 
 words chosen from 20 items . skin temperature was measured using an infrared tympanic probe thermometer , first 
 temp genius ( sherwood medical , crawley , sussex , uk ) . measurements were obtained 
 on the dorsal aspect of the hand in a matrix of five points . the difference in 
 mean temperature between the crps1 and contralateral extremities was 
 calculated , and the data were expressed with respect to the temperature of the 
 unaffected hand . volume was measured with a volumeter , which measures the amount of water displaced by 
 immersion of a body part . the difference between the crps1 and contralateral extremities was calculated 
 as a percentage of the contralateral extremity . mobility was assessed by measuring the active range of 
 motion ( arom ) , which is defined as the arc of motion requiring muscle power to 
 achieve the motion of a joint [ 20 , 21 ] . the 
 arom on the crps1 extremity was multiplied by 100 and divided by the arom on 
 the contralateral extremity to derive the percentage of normal mobility . artificial skin blisters were 
 induced using a suction method [ 9 , 14 , 22 ] . a skin 
 suction chamber was positioned on the skin of the crps1 and contralateral 
 extremities . a vacuum of 300 mm hg was applied with an atmoforte 350 a aspirator 
 pump ( atmos medizintechnik , lenzkirch , germany ) . after 15 minutes , the vacuum was reduced to 250 mm hg , 
 and after another 15 minutes 
 it was reduced to 200 mm hg . the 
 blisters created were punctured , and fluid was pooled from each side into a 1.5 ml 
 eppendorf conical polypropylene tube and centrifuged for 5 minutes at 1600  g. all samples were stored in 1 ml 
 conical polypropylene tubes at 80c until analysis [ 9 , 22 ] . blister fluid samples were diluted 4-fold 
 in appropriate calibrator dilutent assay buffer for the direct measurement of 
 cytokines . cytokine assays were performed following the manufacturer 's protocol , 
 pelikine human elisa kits for il-6 [ m1906 ] and tnf- [ m1920 ] ( clb , amsterdam , 
 the netherlands ) . the standard curve ranges and mean calculated zero signal 3 ( sd ) were 080 and 0.3 pg / ml 
 for il-6 and 01000 and 1 pg / ml 
 for tnf-. the absorbance per well was measured at 450 nm with a medgenix easia 
 reader . sample concentrations were calculated using the appropriate standard 
 calibration lines and the softmax software of the reader . data were analyzed with spss for 
 windows , version 14.0 . to determine whether the 12 crps1 patients from this 
 study were a representative group of the initial 66 crps1 patients who started 
 the follow - up study , the one - way anova test was used for a comparison between 
 groups for the proinflammatory cytokines , and the outcome parameters pain , 
 temperature , volume , and mobility . to determine if the differences in means 
 for these selected parameters were significantly different from 0 , we applied 
 the one - sample t - test for comparisons of the 
 levels of the proinflammatory cytokines between 
 both extremities , and to the differences in the 
 outcome parameters of temperature , volume , and mobility . mixed 
 model analysis was used to compare the differences between both extremities to 
 each other for the levels of the cytokines , vas , mcgill , temperature , volume , 
 and mobility between each time point . correlations 
 between the differences in levels of il-6 and tnf- and the outcome parameters 
 were calculated using the pearson correlation test . significance 
 was accepted at the p < .05 ( two - sided ) level . the group of 12 crps1 patients was a good representation of the original group of 66 crps1 patients 
 with which we initiated the follow - up study 6 years ago . the groups were equal 
 for difference in levels of il-6 ( p = .60 ) and tnf- ( p = .72 ) . they were also equal for the mcgill 
 pain score ( p = .41 ) , 
 temperature difference ( p = .41 ) , 
 volume difference ( p = .48 ) , 
 and mobility ( p = .38 ) . the two groups were only significantly different from each other for vas pain ( p = .03 ) . the characteristics 
 and medication use of the 12 patients with crps1 in one extremity for 6 years ( median ) 
 who were examined 3 times during the follow - up of their crps1 are presented in 
 table 1 . the il-6 and tnf- levels in blister fluid at all time intervals are 
 presented in table 2 . the differences in levels of il-6 between both 
 extremities did not significantly change at t1 compared to t0 ( p = .531 ) and at t2 
 compared to t0 ( p = .063 ) . however , the differences in levels did significantly decrease at t2 compared to 
 t1 ( p = .028 ) . the differences in levels of tnf- between both extremities did not 
 significantly change at t1 compared to t0 ( p = .892 ) and at t2 compared to t0 ( p = .153 ) . the decrease 
 in differences from t1 to t2 almost reached significance ( p = .064 ) . at the t0 and t1 measurement , the difference in 
 levels of il-6 and tnf- between the crps1 side and the contralateral side in 
 blister fluid was significantly different from 0 . however , at the t2 
 measurement , no significant differences between the two sides were evident 
 ( figures 1(a ) and 1(b ) ) for the 12 
 crps1 patients . also the signs and symptoms of impairment were measured at 
 t0 , t1 , and t2 in terms of pain and differences in temperature , volume , and 
 mobility between the crps1 and the contralateral extremities . the vas pain did 
 not significantly change during the course of the disease in these 12 patients 
 ( p = .472 ) . the mcgill pain score improved significantly at t1 
 compared to t0 ( p = .006 ) , 
 but pain was significantly worse at t2 compared to t1 ( p = .041 ) . furthermore , 
 no significant improvement was seen at t2 as compared to t0 ( p = .567 ) . the vas pain 
 and the mcgill pain score significantly differed from 0 during all 6 years of 
 follow - up ( figures 2(a ) and 2(b ) ) . the absolute difference in temperature and volume 
 between both sides did not change during the course of the disease ( p = .204 and p = .509 , 
 resp . ) . the difference between the crps1 and contralateral side did vary 
 significantly from 0 at all three moments of measurement for temperature and 
 volume ( figures 2(c ) and 2(d ) ) . mobility improved significantly at t1 and t2 as compared 
 to t0 ( p = .002 
 for both ) , with no significant increase in arom at t2 compared to t1 ( p = .353 ) . the 
 difference in mobility between both sides was significantly different from 0 at 
 t0 and t1 , but no significant difference was found at t2 ( figure 2(e ) ) . no correlation was found between the differences in levels of 
 the proinflammatory cytokines and the other disease - related parameters pain and 
 differences between both extremities for temperature , volume , and mobility 
 during the course of the disease . the total use of disease - related medication was divided in 8 categories 
 ( see table 1 ) . the total number of patients using medication as well as the variety in 
 types of medication decreased during the course of the disease . in a six years 
 follow - up study in 12 crps1 patients , we found a decrease in the extent of the 
 differences in levels of tnf- and il-6 in blister fluid . after disease 
 duration of 4 months ( t0 ) and of 3 years ( t1 ) , the levels of these cytokines 
 were significantly higher in the crps1 extremity compared to the contralateral 
 extremity . however , after 6 years ( t2 ) the differences in the cytokine levels 
 between the two extremities were not significantly different . in contrast , the difference 
 in mobility between both sides was significantly improved after 6 years . pain 
 and the differences in temperature and volume were not significantly altered 
 during the course of the disease . no correlation was found between the 
 proinflammatory cytokines and the disease characteristics . because 
 tnf- and il-6 are proinflammatory cytokines , one might expect that these 
 markers of inflammation would be directly related to the characteristics of 
 inflammation : pain , temperature increase , edema , and loss of function . in this study , the levels of tnf- and il-6 were 
 diminished during the course of the disease , but no clear improvement of 
 inflammatory signs was found . the 
 vas score did not change during the course of the disease , and while the mcgill 
 pain score improved at first , it worsened again at the third measurement . the 
 absolute temperature difference tended to diminish during the course of the 
 disease ; however , this was not significant . the volume difference also did not 
 change significantly during the course of the disease . only the arom improved 
 significantly after 6 years ; the patients only described some stiffness of the 
 joints . no improvement of the signs and symptoms of 
 inflammation was found as described above , however after six years , a decline 
 in the number of patients using medication that could counteract inflammation 
 has been observed , such as nsaids , opiates , and antioxidants . we concluded that 
 the normalization of inflammatory mediators after six years was not affected through 
 pharmacological intervention , but due to a diminution of disease activity . an explanation for a decrease in differences in the levels of 
 tnf- and il-6 between both sides during the course of crps1 is a spreading of 
 the disease from the crps1 side to the contralateral extremity . this spreading 
 could also be explained by the fact that the levels in the crps1 side are 
 increased during the course of the disease . however , the increased levels of 
 the contralateral side measured 6 years after the initial event do not even 
 come close to the high levels of tnf- and il-6 measured in the crps1 side 4 
 months and 3 years after the initial event . furthermore , the levels of il-6 of 
 both sides at t2 are within the normal range of the levels measured in blister 
 fluid from healthy controls ( mean 16 pg / ml ) . finally , one would expect that the differences between both sides in disease 
 activity would decrease during the course of the disease when there is 
 spreading . this is not confirmed in this follow - up study . in the present 
 study , in contrast with the diminished levels of the proinflammatory cytokines during the course of the disease , signs of inflammation were found in all three 
 stages of the disease . in an earlier study , during the first two years of 
 crps1 , about half of the patients showed no inflammatory signs , but did fulfil the criteria 
 of bruehl et al . . several other studies confirmed that observation , even describing a primarily cold 
 crps1 [ 4 , 10 , 25 , 26 ] . an explanation for finding a temperature increase and decrease already at the 
 onset of crps1 is that blood flow ; and thus changes in skin temperature are 
 the results of not only afferent mechanisms , such as local mediators , but also 
 efferent mechanisms . disturbances in 
 central temperature regulation could result in altered ( local ) temperature of 
 the injured extremity [ 2729 ] . recently , we 
 found evidence that temperature changes also might be partly caused by the crosstalk 
 in the vascular system between higher levels of cytokines and the nitric 
 oxide / endothelin-1 ( no / et-1 ) balance [ 30 , 31 ] . furthermore , blood flow and tissue - blood distribution could be 
 diminished , partly due to disuse of the extremity . a possible explanation for finding edema and atrophy 
 during all stages of the disease is that edema is not only affected 
 by tnf- and il-6 , but also by other inflammatory mediators such as calcitonin 
 gene - related peptide ( cgrp ) and substance p ( sp ) , and related mechanisms [ 32 , 33 ] . no correlation was found between the differences in levels of the cytokines and pain and 
 differences between both extremities for temperature , volume , and arom for all 
 three times of measurement . in an earlier study , we found similar results . perhaps il-6 and tnf- are 
 only partially responsible for the permanent damage in crps1 , expressed as 
 pain , and changes in temperature , volume , and mobility . other mediators or a 
 combination of mediators , such as nitric oxide   and/or amino acids [ 34 , 35 ] or other mechanisms , may also play a role in the 
 pathophysiology of crps1 and may explain in part the course of the disease .
in an earlier study , levels of the proinflammatory cytokines tnf- and il-6 are higher in blisters fluid from the complex regional pain syndrome type 1 ( crps1 ) side obtained at 6 and 30 months ( median ) after the initial event . 
 the aim of this follow - up study is to determine the involvement of these cytokines in long lasting crps1 . 
 twelve crps1 patients , with median disease duration of 72 months , participated . 
 the levels of tnf- and il-6 were measured in blister fluid ; disease activity was reevaluated by measuring pain and differences in temperature , volume , and mobility between both extremities . 
 differences in levels of il-6 and tnf- and mobility between both sides were significantly decreased . 
 pain and differences in temperature and volume were not significantly altered . 
 no correlation was found between the cytokines and the disease characteristics . 
 these results indicate that il-6 and tnf- are only partially responsible for the signs and symptoms of crps1 .