Journal of Pain Research (Nov 2022)
The Endocannabinoid Analgesic Entourage Effect: Investigations in Cultured DRG Neurons
Abstract
Uma Anand,1 Barbara Pacchetti,2 Praveen Anand,3 Mikael Hans Sodergren1,2 1Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, London, W12 0HS, UK; 2Curaleaf International Limited, London, EC2A 2EW, UK; 3Professor of Clinical Neurology, Department of Brain Sciences, Imperial College London, London, W12 0HS, UKCorrespondence: Uma Anand, Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0HS, UK, Tel +44 020 3313 2362, Fax +44 020 3313 3363, Email [email protected]: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the “entourage effect”. We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors.Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 μM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 μMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3– 7 rats, controls N=16), and analysed.Results: In controls, 1 μMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10– 100 μMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001– 100 μMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P< 0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 μMol (P< 0.001), in 60% neurons, as did 2-LG at 0.1– 100 μMol (P< 0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 μMol 2-AG, 5 μMol 2-PG and 10 μMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P< 0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist.Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.Keywords: entourage effect, endocannabinoids, DRG neurons, nociception, analgesia
