Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear. Inflammatory cells, in particular macrophages, are critical components of the response to nerve injury. Using ultrasmall superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI) to monitor macrophage trafficking, the purpose of this project is to determine whether minocycline modulates macrophage trafficking to the site of nerve injury in vivo and, in turn, results in altered pain thresholds.
Animal experiments were approved by Stanford IACUC. A model of neuropathic pain was created using the Spared Nerve Injury (SNI) model that involves ligation of the left sciatic nerve in the left thigh of adult Sprague–Dawley rats. Animals with SNI and uninjured animals were then injected with/without USPIOs (300μmol/kg IV) and with/without minocycline (50mg/kg IP). Bilateral sciatic nerves were scanned with a volume coil in a 7T magnet 7days after USPIO administration. Fluid-sensitive MR images were obtained, and ROIs were placed on bilateral sciatic nerves to quantify signal intensity. Pain behavior modulation by minocycline was measured using the Von Frey filament test. Sciatic nerves were ultimately harvested at day 7, fixed in 10% buffered formalin and stained for the presence of iron oxide-laden macrophages. Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p<0.011). Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/−0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/−4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/−2.3%) (p<0.04). Histology of harvested sciatic nerve specimens confirmed the presence USPIOs at the nerve injury site in the SNI group without minocycline treatment.
Animals with neuropathic pain in the left hindpaw show increased trafficking of USPIO-laden macrophages to the site of sciatic nerve injury. Minocycline to retards the migration of macrophages to the nerve injury site, which may partly explain its anti-nociceptive effects. USPIO-MRI is an effective in vivo imaging tool to study the role of macrophages in the development of neuropathic pain.
Keywords: Neuropathic pain, Macrophages, Magnetic resonance imaging, Iron-oxide nanoparticles, Spared-nerve injury model