ENDOCANNABINOID CEREBROSPINAL FLUID LEVELS IN MIGRAINE 87 4 Discussion Here we measured and compared concentrations of the eCBs AEA, 2-AG, and DHEA in CSF of 194 individuals with migraine with (n = 97) or without aura (n = 97) in the interictal state and 94 healthy controls. The three study populations were matched for age and sex. The CSF sampling and processing were strictly protocoled, and the analysis was performed with our validated microliquid chromatography – tandem mass spectrometry quantification (micro-LC-MS/MS).31 We found no differences in eCBs related to migraine status. However, covariates age, sex, daily number of cigarettes, and BMI had an effect on the different eCB concentrations. The effect on the covariates emphasizes the complexity of the endocannabinoid system regarding confounding factors. Interestingly, 2-AG levels were positively correlated with lifetime depression, which has implications for further research as comorbidity with depression in migraine patients makes them more prone for chronification and medication overuse.35 So far, only two studies have assessed the levels of eCBs in body fluids of individuals with migraine, but the results were contradictory.20, 36 In one study, concentrations of AEA were lower and those of the eCB analogue PEA were higher in the CSF of 15 individuals with chronic migraine versus 20 age-matched controls.20 However, in that study, the analysis of eCBs was based on a different extraction method (i.e., the Bligh and Dyer liquid-liquid extraction method), which may generate AEA during the analytical step (see e-Figure 1, supplementary material). Most importantly, there was no correction for confounding factors as we did in our study. Therefore, the results of that study should be interpreted with caution. In the other study, 16 different eCBs were measured by LC-MS/MS in blood plasma from 38 females with episodic migraine between attacks and 26 healthy controls collected on days 1 and 14 of the menstrual cycle.36 There were no differences for AEA and N-acylethanolamines. In addition, in a subset of study participants eCBs were measured in plasma collected during attacks and compared with the interictal measurement; again no differences were found.36 In both studies, the number of participants was limited, the body fluids in which the measurements were done were different, the sampling conditions were not protocoled, and no correction for all possible confounding factors was done. Contrary to the previous suggestion of the influence of endocannabinoids in migraine pathophysiology, no association between the endocannabinoid system and migraine was detected in our CSF study.9, 20-22 A dysregulation of the endocannabinoid system might have remained undetected in case it involves only a localized (e.g., near the synapse) or migraine-attack related disturbance of the endocannabinoid system and not a generalized phenomenon that shows in CSF. Basal levels of eCBs in the brain are low, with 2-AG levels much higher than those of AEA in the brain.37 This may be an explanation for not finding differences in CSF in migraine patients in the interictal phase. Further limitations of our study are that due to the size of the study and the great care taken to match our study groups, including for age, sex and diurnal and seasonal time of sampling, the samples were collected over a fairly long time. This could have potentially
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