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Development of a method for the measurement of endocannabinoid release in the rat brain by microdialysis Substantial levels of endocannabinoids (ECs) have been measured in brain tissues. However, the measurement of extra-cellular levels of ECs in brain regions in vivo has received little attention due to a lack of a sensitive and specific assay to detect these lipophilic compounds using microdialysis. The present study reports the development of an appropriate liquid chromatography-tandem mass spectrometry analytical method that can be used in conjunction with in vivo microdialysis. Statistical analysis was performed using Students t-test or one way analysis of variance as appropriate. Optimal in vitro recovery of ECs was obtained with a polycarbonate microdialysis probe, CMA12, 20 KD cut-off, compared to other probe materials including polyacril nitrile, cuprophane, polyethyl sulphone, regenerated cellulose and copolymer of acrilonitrile-sodium metallyl sulfonate. Recovery of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) standards was improved (35% and 45%, respectively) by the addition of 20% hydroxy-propyl β-cyclodextrin (HP-β-CD) to the perfusion fluid (artificial Cerebrospinal fluid; aCSF) while addition of BSA (4%) had no effect. Collection of dialysate standards in HPLC glass vials improved the recovery of ECs by 2.5±0.3 fold (20-35% recovery; mean ± s.e.m.) compared to the use of plastic microtubes or silanised glass insert vials. Liquid-liquid extraction of dialysates in order to minimize the salt content and to remove HP- β-CD was necessary to avoid producing a damaging precipitation within the spectrometer and to prevent secondary elution of ECs from the HP- β-CD. Extraction of standards with ethyl acetate/hexane (9:1) and reconstitution in acetonitrile successfully removed HP- β-CD and significantly (n=6, P<0.0001) enhanced AEA and 2-AG recovery by 2±0.4 fold, oleoyl ethanolamide (OEA) by 3.2±0.6 fold (n=6, P<0.0001) and palmitoyl ethanolamide (PEA) by 2.3±0.2 fold (n=6, P=0.044) (mean±SEM)compared to extraction with acetonitrile or evaporation of dialysates and reconstitution in acetonitrile. Although HP- β-CD improved in vitro recovery of standards, the relatively high viscosity of the 20% perfusion solution inhibited flow through dialysis tubing. To combat this problem, a range of different cyclodextrins was investigated. Compared with 3 other cyclodextrins (β-CD, β-CD hydrate and α-CD) methyl-β-CD possesses high solubility in water (up to 30%) with low viscosity, allowing an acceptable flow rate during microdialysis. Methyl-β-CD (5%) increased AEA recovery by 3.3±0.3 fold (n=6, P=0.0001) and 2-AG by 3.7±0.6 fold (n=6, P<0.0001). In preliminary experiments, in vivo microdialysis was performed on male Lister hooded rats (250-300 g). Probes were implanted under isoflurane anaesthesia and dialysates were collected 24h later in the freely moving animal. In a single experiment in the striatum, with 20% HP-β-CD included in the aCSF at a flow rate of 2μ /min, basal levels of AEA 0.8 pmol/ml and OEA 1.4 pmol/ml were detected which increased to 9.6 pmol/ml and 14.0 pmol/ml respectively by perfusing the depolarising agent veratridine (50 μM) through the probe. In one additional striatal study, basal levels of AEA 2.1 pmol/ml and OEA 4.1 pmol/ml were detected which increased following injection of the dopamine D2/D3 receptor agonist quinpirole (2.5 mg/kg i.p) to 11.5 and 28.0 pmol/ml respectively. In the hippocampus, only OEA (3.9±0.7 pmol/ml) and PEA (4.2±0.5 pmol/ml) were detected and perfusion of 100mM KCl increased the levels to 5.6±1.1 fold (n=18, P=0.0007) and 9.1± 2.7 fold (n=18, P<0.0001) respectively. The method developed is capable of detecting the release of some, but not all, ECs in rat brain regions. Further refinement is required to facilitate a more complete characterisation of EC release by microdialysis including application of nano-spray technology. |
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