Background and Aim:

Central neuropathic pain (CNP) is a common chronic complication following spinal cord injury (SCI) that significantly reduces the quality of life: current treatments for CNP have limited efficacy and side effects. Activation of microglia in response to nerve injury contribute to the pathogenesis of CNP1. We have previously shown that docosahexaenoyl- (DHEA) and eicosapentaenoyl-ethanolamine (EPEA), which are omega-3 long chain polyunsaturated fatty acids, both alone and in combination inhibit microglial activation2,3. Since microglia express cannabinoid receptors (CB1 and CB2) and DHEA and EPEA are considered to be endocannabinoids4, we aimed to investigate if these fatty acids are ligands for CB1 and CB2 and to further dissect their mechanism of action.

Methods:

HEK293 cells were transfected with CB1-Rluc8 or CB2-Rluc8, Gαi and β-arrestin and BRET in the presence of Coelenterazine (1mM) using a Clariostar plate reader (BMG Labtech) +/- DHEA or EPEA.  Phosphorylation of extracellular signal-regulated kinases (ERK1/2) was determined by western blot in CB1- and CB2-HEK293 in response to DHEA or EPEA +/- pertussis toxin. Primary microglia (Sprague Dawley rat, 3-5 postnatal) were isolated and treated with LPS (100ng/ml) +/- DHEA and/or EPEA and qPCR and RT2 Profiler PCR Arrays (Qiagen) were used to measure the mRNA expression of proinflammatory mediators over time. Statistical comparisons were performed using unpaired Student's t-test or one-way/two-way ANOVA, with p <0.05 considered significant.

Results: 

DHEA and EPEA does-dependently increased CB1- and CB2-dependent Gαi and β-arrestin activity. Furthermore, DHEA and EPEA significant increased phosphorylation of ERK1/2 in CB2-HEK293, which was pertussis toxin sensitive. QRT-PCR showed both DHEA and EPEA blunted LPS-mediated increase in IL-6 and IL-1β, which peaked at 6 hrs, over 50%.

Conclusion:

Taken together these data show DHEA and EPEA, which are anti-inflammatory in microglia, act, at least in part via CB1- and CB2-signalling. DHEA and EPEA could be novel therapeutic options for managing SCI-CNP.

1.         Finnerup NB, Baastrup C. Spinal cord injury pain: Mechanisms and management. Curr Pain Headache Rep. 2012;16(3). doi:10.1007/s11916-012-0259-x

2.         Georgieva M, Wei Y, Dumitrascuta M, Pertwee R, Finnerup NB, Huang W. Fatty acid suppression of glial activation prevents central neuropathic pain after spinal cord injury. In: Pain. Vol 160. ; 2019. doi:10.1097/j.pain. 2724-2742

3.         Abdallah A, Wei Y, Pertwee R, McCaig C, Huang W. The Potential of Omega-3 Endocannabinoids for Managing Central Neuropathic Pain After Spinal Cord Injury International Association for the Study of Pain. Conference; 2022.

4.         Brown I, Cascio MG, Wahle KWJ, et al. Cannabinoid receptor-dependent and -independent anti-proliferative effects of omega-3 ethanolamides in androgen receptor-positive and -negative prostate cancer cell lines. Carcinogenesis. 2010;31(9). doi:10.1093/carcin/bgq151