Unique to sickle cell disease (SCD) are unpredictable episodes of acute pain leading to hospitalization and reduced survival. These are believed to be due to vasoocclusion, but the underlying mechanisms are not understood. Circulating sickle red blood cells (RBCs) can adversely affect the organs in the periphery as well as the central nervous system (CNS). Thus, we examined the peripheral and central mechanisms of acute pain using a model of vasoocclusion caused by hypoxia/reoxygenation (H/R) (8% O₂ for 3 h followed by reoxygenation @ room air) in homozygous HbSS BERK sickle and HbAA BERK control mice. Earlier studies from our laboratory demonstrated increased inflammation, oxidative stress and p38 MAPK (p38) activation in the periphery (dorsal root ganglion, DRG) and the CNS (spinal cord) of sickle mice compared to control. H/R further increased levels of p38 phosphorylation, interleukin-17 (IL-17), tumor necrosis factor-alpha (TNF-a), and interleukin-6 (IL-6) significantly (P<0.05) in the spinal cords of sickle mice compared to normoxia. We hypothesized that IL-17 induces TNF-α, IL-6, and p38 phosphorylation, leading to oxidative stress and neuronal damage resulting in severe pain. We administered anti–IL-17A antibodies (Ab, 0.25 μg/g body weight; #A2025, Selleck Chemicals, Houston, TX) intrathecally in the spinal cord 4 h before inciting H/R twice (H/R-1 and H/R-2) at 48 h interval. Vehicle-treated sickle mice showed increased mechanical and cold sensitivity at all post-H/R time points (H/R-1, 24 h post-H/R-1, H/R-2, 24 h post-H/R-2) vs. baseline (BL) and control mice (P<0.0001). Grip force (GF) declined after H/R-1 (P<0.05) and further after H/R-2, indicating increased musculoskeletal hyperalgesia compared to control mice (P<0.0001). Anti-IL-17 Ab significantly reduced mechanical (P<0.0001), thermal (P<0.0001), and musculoskeletal hyperalgesia (P<0.05) compared to vehicle treated sickle mice at respective time points after H/R-1 and H/R-2. The analysis of sickle mice post-H/R-2 showed decreased IL-17 and TNF-α in the spinal cords of sickle mice treated with anti-IL-17 Ab compared to vehicle, suggesting that spinal IL-17 plays a critical role in evoking acute hyperalgesia by regulating inflammatory cytokines. We next examined if the novel transdermal curcumin (TDC; Vasceptor, Vascarta Inc.), which reduced inflammation and oxidative stress in sickle mice in the periphery (Goel et al., PNAS nexus 2025), has the potential to inhibit spinal inflammation, oxidative stress, and p38 phosphorylation. After baseline (BL) measures of hyperalgesia, mice received daily TDC or vehicle for 2 weeks, followed by 2 treatments of H/R-1 and H/R-2 to elicit acute pain. TDC lowered mechanical and cold sensitivity, 24 h post-H/R-1, H/R-2, and 24 h post-H/R-2 vs. vehicle (P<0.0001). GF increased significantly after H/R-1 and HR2 compared to respective vehicle treated mice (P<0.05). Thus, pre-treatment with TDC significantly reduced H/R-evoked hyperalgesia. Analysis of spinal cords from TDC and vehicle treated mice following H/R-2 showed a decrease in multiple cytokines, including IL-17, IL-6 and TNF-α, similar to a decrease observed with spinal anti-IL-17 Ab. Since p38 is activated by cytokines and is also involved in the transcriptional regulation of cytokines, leading to oxidative stress, we next examined if TDC or inhibition of p38 with neflamapimod (Nef) would prevent hypoxia-induced oxidative stress in a sickle microenvironment in the peripheral and CNS. We found that primary DRG neurons from sickle mice and HT22 hippocampal neurons exposed to TNF-α + hemin ± CoCl₂-induced hypoxia demonstrated marked reactive oxygen species (ROS) generation and mitochondrial depolarization (P<0.05 to P<0.0001), consistent with SCD pathophysiology. The p38 inhibitor, Nef reduced ROS in HT22 (P<0.0001) and DRG neurons (P<0.001) and stabilized mitochondrial potential (P<0.0001), confirming p38 as a central mediator. Similarly, TDC reduced ROS in both HT22 and DRG neurons under sickle microenvironment/hypoxia (P<0.001) and preserved mitochondrial membrane potential (P<0.05 to P<0.0001) compared to vehicle. We conclude that IL-17 orchestrates acute pain by promoting IL-17-TNF-α/IL-6–p38 cascade in the CNS and periphery, leading to oxidative stress and mitochondrial dysfunction in sensory neurons. TDC interrupts this SCD pathophysiological cascade, suggestive of a preventive effect on acute pain episodes in SCD.

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2025
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