Evans Blue Reduces Neuropathic Pain Behavior by Inhibiting Spinal ATP Release

Abstract

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1β and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL.