ALBERT

Description: Sickle cell disease (SCD) is associated with pain, which remains a major challenge to treat. Earlier, we showed that peripheral mechanisms including mast cell activation in the skin contribute to pain in sickle mice (Vincent et al., Blood 2013). Mast cell activation in sickle mice was accompanied by a significant increase in toll-like receptor 4 (TLR4) as compared to mast cells from control mice. Since peripheral as well as central mechanisms are involved in nociception, we examined the central mechanisms underlying pain in SCD. TLR4 signaling is involved in inflammatory and neuropathic pain (Wang et al., FASEB 2013 and Hutchinson et al., Eur J Neurosci 2008). Microglial cells, the “macrophages” of the central nervous system in the spinal cord are critically involved in the development and maintenance of pain. Binding of an endogenous ligand to TLR4 is an important step in the regulation of microglial activity in pain facilitation. We hypothesized that heme, released during hemolysis in SCD, is a ligand for TLR4 expressed on spinal microglia. Methods. We isolated microglial cells from the spinal cords of HbSS-BERK (sickle) and HbAA-BERK (control) mice. To assess mitochondrial activity, we analyzed reactive oxygen species (ROS) and ATP, since increased ROS and decreased ATP are suggestive of mitochondrial dysfunction, which in turn is influenced by endoplasmic reticulum (ER) stress. ROS in the microglial cells was determined by utilizing the cell permeable reagent 2’,7’-dichlorofluorescein, which is oxidized by ROS to form a fluorescent compound, with the max excitation and emission spectra of 495 nm and 529 nm, respectively. ATP production was measured by a luminescence based assay from PerkinElmer (ATPlite). Results. Stimulation of microglia from control and sickle mice with hemin in vitro led to a several-fold increase in TLR4 gene transcripts in a time-dependent manner. Additionally, hemin induced the production of proinflammatory cytokines, TNF-α and IL-6, and ROS compared to vehicle-treated microglial cells from both sickle and control mice (p