Mechanisms Underlying Acute Pain in Sickle Cell Disease

In sickle cell disease (SCD) recurrent and unpredictable episodes of acute pain lead to hospitalization, opioid use, reduce survival and impair the quality of life. It is believed to be caused by occlusion of blood vessels with sickle red blood cells and other cells leading to ischemia/reperfusion (IR) injury, oxidative stress and inflammation. It is an understatement to say that how this occlusion leads to acute pain remains uninvestigated. Hypoxia/reoxygenation (HR) has been used to examine episodes of acute pain/hyperalgesia to simulate vasoocclusive crisis (VOC) in mouse models of SCD. We examined the nociceptive mechanisms triggered by HR in humanized BERK sickle (HbSS) and control BERK mice expressing normal human HbA (HbAA). HR was incited in ~3-month-old female mice by 2 repeated exposures at 48-hour intervals (HR1 and HR2) of 3 hours of hypoxia with 8% O₂ and 92% N₂ followed by reoxygenation for 1 hour in room air, and a group of mice were kept under normoxia. At <3 months of age BERK HbSS do not show constitutive hyperalgesia, which usually develops at ~3.5 months of age. Mechanical, cold and deep tissue hyperalgesia was analyzed before HR, 1 and 24 hours after HR1, and 1 hour after HR2. At baseline (BL) before HR incitement HbSS and HbAA mice showed no hyperalgesia. We observed a significant increase in mechanical and cold hyperalgesia in HbSS mice after 1 hour of HR1, which was sustained until 1-hour post-HR2 (p<.01 vs. BL); and was significantly higher than mice under normoxia at each time point post HR1 and 2 (p<.01). HbAA mice did not show an increase or decrease in hyperalgesia following both HR1 and 2 compared to their BL. Mice were euthanized following 1 hour of HR2 and blood and spinal cords were collected for analysis. Complete Blood Counts showed a significant decrease in MCHC (p<.01) and an increase in platelets (p<.001) following HR2 vs. normoxia in HbSS mice. Analysis of the HbSS mice spinal cords using a multiplex cytokine array showed a significant increase in multiple cytokines following HR2 vs normoxia, which include, IL1α (p<.000), IL1β (p<.01), IL2 (p<.05), IL3 (p<.01), IL4 (p<.001), IL5 (p<.000), IL6 (p<.05), IL10 (p<.05), IL12 (p<.01), IL17 (p<.001), IFNγ (p<.01), MIP1α (p<.000), TNFα (p<.01) and RANTES (p<.01). All these spinal cytokines were also significantly higher in HbSS vs. HbAA post HR2 (P<.000 to .05 for each). Malondialdehyde, a marker of oxidative stress and end product of membrane lipid peroxidation was significantly increased in HbSS following HR2 vs. normoxia (p<.03), suggestive of oxidative stress, membrane damage and cellular injury. Membrane attack complex (MAC) assembly with C5b-9 leads to pores in the membrane and cellular injury. We found that complement C5b-9 was significantly increased in the plasma of HbSS mice post HR2 vs normoxia (p<.02). Cytokines such as IL1α can activate Complement system via activation of p38 involved in initiation and maintenance of pain. In turn, Complement system can activate p38, leading to a vicious cycle of their sustained activation and cell damage. We found that p38 phosphorylation was increased ~3-fold (p<.05) throughout the cell body and ~2-fold (p<.02) in nuclear translocation in the spinal cords of HR treated HbSS vs. normoxia. Thus, p38 phosphorylation may be transcriptionally regulating the activity of cytokines as well as the membrane bound MACs. These data suggest that IR during VOC stimulates a highly pro-nociceptive milieu of inflammation and cellular injury in the central nervous system which may lead to complex inflammatory and neuropathic pain. The second order neurons in the spinal cord play a critical role in processing and transmitting the nociceptive signals to the brain for pain perception. Spinal cord is highly vascularized. It is likely that hypoxia leads to initial injury by disrupting blood-spinal barrier via activation of MAC, which is sustained/enhanced by the activation of p38 signaling leading to the release of multiple cytokines, continued vascular disruption, heme-induced ferroptosis via IL17, oxidative stress and continued pain, which may even lead to the transition from acute to chronic pain. Our data show for the first time the mechanisms underlying acute VOC pain. Strategies targeting multiple cytokines, their receptors and pathways using combination therapy have shown promise in the treatment of complex chronic pain etiologies and may be potentially beneficial in targeting acute pain in SCD.