Potential Application of Boldine in Alleviating SCI-induced Neuropathic Pain

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Spinal cord injury (SCI) affects over 291,000 Americans of whom ~47,000 are veterans. Providing high quality care to veterans with SCI is a priority within the Department of Veterans Affairs. For SCI patients, pain is one of the most difficult complications to treat, and this is especially true for neuropathic pain (NP) which is known to be chronic and refractory to many currently available treatments. Until now, there has been no fully effective therapy for neuropathic pain in SCI. Recent studies have shown that after nerve injury, Toll-like receptors (TLRs) are involved in the generation of neuropathic pain. Our feasibility studies in primary astrocytes suggested that boldine, a naturally occurring compound, could inhibit the TLR-inflammasome signaling. Pilot studies conducted in a mouse model of moderate contusion SCI revealed that boldine improved locomotor function following SCI and reduced the expression of major components of the TLR family and inflammasome complexes in brain regions linked to pain processing. Based on these preliminary findings and the important role of toll-like receptors and inflammasome complexes in neuropathic pain, we propose to conduct preclinical studies using sensory functional outcome measurements and molecular mechanistic studies to test whether oral administration of boldine will alleviate pain responses in a mouse model of SCI. Outcomes will support follow-up preclinical and clinical translational studies for potential application of this natural compound in reducing neuropathic pain in veterans with SCI. Aim 1. Test the effect of boldine on regulating TLR and inflammasome pathways in primary cultures. Rationale: TLRs are responsible for the stimulation of astrocytes and microglia and will induce expression in these cells of proinflammatory mediators and cytokines, therefore leading to the generation and maintenance of neuropathic pain. Our preliminary findings in mouse primary astrocyte cultures suggested that boldine treatment significantly reduced the expression of some major components in the TLR-inflammasome pathways, including TLR2, NFκB, Caspase-1 and IL-1β. Hypothesis: Boldine will inhibit TLR-inflammasome pathway in vitro. Methods: Mouse primary astrocyte, microglia, dorsal horn and DRG cultures will be treated with LPS in the presence or absence of 25 or 50 µg/ml boldine. Cytotoxicity will be determined by LDH assay. The mRNA and protein levels for major components of the TLR-inflammasome will be determined by qPCR or western blot, respectively. IL-1β level in the medium will be measured by ELISA, NFκB nuclear localization will be determined by immunofluorescence, and Caspase-1 activity will also be measured. Aim 2. Determine the effects of boldine on reducing neuropathic pain in a mouse model of moderate severity contusion SCI and dissect possible mechanisms. Rationale: Spinal cord injuries in the mice have been a widely used model to evaluate different experimental treatment strategies. Our preliminary findings in a mouse model of moderate contusion SCI suggested that administration of boldine improved locomotor function following SCI and reduced the mRNA levels of several major components of the TLR family and inflammasome complexes in the brain. Hypothesis: Boldine will reduce SCI-induced neuropathic pain through normalizing the TLR-inflammasome signaling. Methods: C57BL/6 mice will undergo either a laminectomy or moderate contusion injury at T9-10. Animals will be randomized among 4 groups: 1) Laminectomy, 2) Laminectomy plus boldine, 3) SCI, 4) SCI plus boldine. Boldine will be administered twice daily by gavage to reach a total dose of 50 mg/kg/day. Animals will be evaluated for locomotor function before surgery then weekly thereafter using the Basso Mouse Scale (BMS) method. Von Frey filament test and tail-flick test will be performed before surgery for baseline studies and weekly after injury till 28 dpi. Animals will then be euthanized and brain, spinal cord, dorsal root ganglia (DRG) and serum will be collected for mechanistic studies including qPCR, western blot, immunostaining, Luminex multiplex assay to evaluate the potential regulation of TLR-inflammasome signaling pathways.