In Vivo Negligible Depletion SPME for the Determination of Free and Total Concentrations of Anandamide and 2-Arachidonoylglycerol in the Brain of a Parkinson’s Disease Rat Model

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder, and diagnosis typically occurs after substantial neuronal loss, underscoring the need for biomarkers capable of detecting early pathological changes. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been proposed as potential PD biomarkers; however, only their free fractions are biologically active. In this study, an in vivo solid-phase microextraction (SPME) method was developed to quantify free and total concentrations of AEA and 2-AG in rat brain under conditions of negligible depletion. Under these conditions, the SPME probe functions analogously to a sensor, extracting only minute amounts of analyte without perturbing the equilibrium between free and matrix-bound species. Extractions were performed at equilibrium, and experimentally determined distribution constants in PBS were used to calculate free concentrations. Matrix-binding percentages exceeded 99% for both analytes and were used alongside with free concentration measurements to estimate total concentration levels. In vivo analyses in a 6-OHDA rat model of PD revealed significant elevated striatal AEA levels in lesioned animals relative to controls, supporting its potential as a biomarker of early neurochemical alterations. In contrast, ex vivo SPME extractions in dissected brain did not show increased AEA levels in the PD group, indicating loss of physiologically relevant information post-mortem. Although 2-AG was not detected in vivo, it was quantified ex vivo, suggesting limited active release under the examined conditions. Overall, these findings highlight the capability of in vivo SPME to capture changes in heavily bound hydrophobic neurochemicals, thereby supporting its application in studies of endocannabinoid dysregulation.