Spinal glial activation and oxidative stress are alleviated by treatment with curcumin or coenzyme Q in sickle mice

Sickle cell anemia (SCA) is accompanied by unpredictable episodes of recurrent acute pain during vaso-occlusive crises (VOC), superimposed on chronic pain.1 Pain in SCA can start in infancy and may continue throughout life, leading to sustained activation of the nociceptive mechanisms resulting in poor therapeutic outcomes. Pain is an outcome of nociceptive processing in the central nervous system (CNS), triggered by peripheral nervous system response to exogenous and endogenous stimuli. Activation of transient receptor potential vanilloid 1 (TRPV1) channels on C-fibers, neurogenic inflammation, mast cell activation, systemic inflammation, and oxidative stress in the periphery have been demonstrated in SCA.32 However, the extent and mechanisms of CNS involvement remain unknown in SCA. The activation of inflammatory and neuronal cells in the CNS plays a pivotal role in nociception.4 We recently observed that spinal nociceptive neurons are sensitized in sickle mice, suggestive of central sensitization.5 Bidirectional signaling occurs between neurons and immunocompetent cells present in the CNS, including microglia, astrocytes and oligodendrocytes.4 Activated microglia release reactive oxidative species (ROS), inflammatory cytokines, neurotrophic factors, and prostaglandins that excite nociceptive neurons and contribute to the persistence of chronic pain.4 It is therefore likely that the activation of central nociceptive mechanisms contributes to chronic pain in SCA.

Remarkable decreases in inflammation, thiobarbituric acid reactive substances (TBARS, an indicator of oxidative stress), and VOC have been observed in SCA patients receiving coenzyme Q10 (CoQ10).6 Additionally, curcumin reduced markers of oxidative stress in thalassemia patients and also ameliorates pain hypersensitivity in rats with monoarthritis by decreasing spinal neuroinflammation.87 Since excess free iron due to hemolysis contributes to oxidative stress and inflammation in SCA, we examined glial activation, inflammation and oxidative stress in the spinal cords of sickle mice and tested the possibility of a synergistic effect of CoQ10 and/or curcumin to ameliorate spinal oxidative stress, glial activation and hyperalgesia.

To examine our hypotheses, we used female transgenic HbSS-BERK sickle mice with murine α and β globin knockouts and expressing human sickle, or normal haemoglobin A (designated sickle or control mice, henceforth, respectively). We bred and characterized these mice by pheno- and genotyping (see Online Supplementary Appendix for details).93 These sickle mice have severe hematologic disease, organ damage and tonic hyperalgesia similar to that observed in human SCA.119

Sickle mice received either vehicle (olive oil), curcumin (15 mg/kg), CoQ10 (45 mg/kg), or both CoQ10 and curcumin (cotreatment) daily for 4 weeks by gavage. Female mice were used because BERK female mice show more hyperalgesia as compared to males.11 Pain behaviors were evaluated during the proestrous/estrous cycle before treatment and weekly. The mice were tested for mechanical hyperalgesia using paw withdrawal frequency (PWF) in response to von Frey filaments, paw withdrawal latency (PWL) in response to a heat stimulus using a Hargreave’s apparatus; and sensitivity to cold was determined by PWL and PWF per 2 min on a cold plate (see detailed procedures in the Online Supplementary Appendix).11 Spinal cords were harvested after 4 weeks of treatment. Sections were examined by laser scanning confocal microscopy (LSCM) for Iba1, a microglial marker (Wako, Richmond, VA, USA), glial fibrillary acidic protein (GFAP), an astrocyte marker (Abcam, Cambridge, MA, USA), neuropeptide substance P (SP, Abcam) and detection of ROS with dihydroethidium (DHE, Life Technologies, Grand Island, NY, USA).