Targeting Central Mechanisms of Pain to Improve Acupuncture Analgesia in a Humanized Mouse Model of Sickle Cell Disease

Background: Pain is one of the major comorbidities of sickle cell disease (SCD), which largely remains reliant on opioid use for analgesia. Side effects of opioids including, but not limited to fear of addiction, constipation, pruritus and opioid-induced hyperalgesia warrant the need for analgesic therapies devoid of side effects. Non-pharmacological strategies including acupuncture have been effective in pain treatment. A retrospective analysis (n=24 patients) showed that acupuncture reduced pain in a majority (75%) of SCD patients (Lu K et al., Clin J Pain. 2014). In a mouse model of SCD, electroacupuncture (EA) on conscious free-moving mice led to variable analgesic response ranging from high- (nociceptive threshold increase >200%), moderate- (threshold between 100~200%) to non-responders (threshold increase ≤100%) (Wang Y et al., Sci Rep. 2016). Substance P (SP), a proinflammatory vasoactive neuropeptide in the periphery and centrally and spinal activated p38 mitogen activated protein kinase (MAPK), critical mediators of chronic pain were significantly increased in sickle mice with moderate or no response to EA analgesia. Increased circulating SP has been reported in SCD patients at steady state and during chronic pain. We hypothesize that chronic pain in moderate- and non-responders is due to central sensitization mediated by SP-induced p38 MAPK phosphorylation; and that inhibiting the effect of SP and/or downstream p38 MAPK signaling would improve response to EA in moderate and non-responsive sickle mice.

Methods: HbSS-BERK sickle mice expressing human sickle hemoglobin without any treatment and those showing moderate- (threshold between 100~200%) and no-response (threshold increase ≤100%); and HbAA-BERK control mice that express normal human hemoglobin A were used. All groups included mice of both genders at 5-7 months of age and were treated daily with 10 mg/kg, i.p. netupitant (antagonist of neurokinin 1 receptor, a receptor for SP), or SB203580, a p38MAPK inhibitor, with or without four sequential EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Hyperalgesia was evaluated daily before starting the inhibitor/EA treatment (baseline, BL) and after treatments throughout 12 days by determining the sensitivity to mechanical-, thermal- and deep tissue-stimuli using von Frey filaments, Hargreaves test, cold plate and grip force, respectively.

Results: Sickle mice showing no- or moderate responsive to EA did not demonstrate a significant effect of netupitant or SB203580 without EA on hyperalgesia. However, co-treatment with netupitant and EA reduced mechanical, thermal and deep tissue hyperalgesia through the entire treatment, reaching significance at day 9 and/or day 12. Co-treatment with netupitant enhanced analgesia of EA by significantly decreasing mechanical hyperalgesia (p<0.05 vs untreated sickle or moderate-responder + netupitant) and heat sensitivity (p<0.05 vs BL) at day 9, cold sensitivity (p<0.05 vs BL) at day 12 for both moderate- and non-responders. Deep tissue hyperalgesia for co-treated moderate-responders (p<0.05 vs BL) was significantly reduced at day 9, while the reduction observed with co-treated non-responders (p<0.05 vs BL) only reached significance on day 12. These data suggest that SP mediates sustained chronic pain in SCD, which imparts resistance to EA analgesia. Co-treatment with SB203580 and EA together decreased heat sensitivity (p<0.05 vs BL) at day 9, mechanical hyperalgesia (p<0.05 vs BL or moderate-responder + EA) and cold sensitivity (p<0.05 vs BL) at day 12 and deep tissue hyperalgesia for moderate-responders (p<0.05 vs BL) at day 9 and 12 and non-responders (p<0.05 vs BL) at day 12. Therefore, central sensitization with increased p38MAPK phosphorylation perhaps mediated by high SP levels prevents the responsiveness to EA.

Conclusion: Increased SP-induced spinal p38MAPK activation may underlie poor responsiveness to EA and perhaps other analgesic therapies in SCD. Therefore, inhibition of SP or p38 MAPK may improve analgesic outcomes with EA. Circulating SP levels may also be predictive of response to EA and/other analgesic therapies. Co-treatment strategies using acupuncture with mechanism-based targets of central sensitization may be potentially beneficial in treating pain and reducing opioid use in SCD.