Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.spinal nerve transection | sulforaphane D amage to the peripheral nerve often results in abnormal chronic pain and pain hypersensitivity that are generally referred to as "neuropathic pain." It is known that hypersensitivity of pain-transmission neurons in the spinal cord is involved in the development of neuropathic pain, and this process has been referred to as central sensitization (1). Various pathological events are reported to precede central sensitization. These include, but are not limited to, p38 activation in spinal cord microglia and subsequent proinflammatory cytokine expression, NMDA receptor phosphorylation in spinal cord neurons, and altered expression of ion channels on neurons (2-5). In addition, a series of studies have implied that production of reactive oxygen species (ROS) in the spinal cord is involved in the induction of neuropathic pain. For example, systemic injection of ROS scavengers, such as phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethylpyrroline-N-oxide (DMPO), relieved spinal nerve ligation-induced pain hypersensitivity in rats (6). In addition, spinal nerve ligation-induced phosphorylation of the NMDA receptor subunit 1 (NR1) in dorsal horn neurons was attenuated by ROS scavengers (7). These results suggested that ROS are critically involved in the development and maintenance of neuropathic pain; however, the exact primary source of ROS production has remained elusive.Recently, a study using the mitochondrial ROS detector MT-Red showed that ROS are generated in the mitochondria of dorsal horn neurons (8). Production of mitochondrial ROS by intrathecal injection of the electron transport complex inhibitors...

BackgroundWe have previously reported that nerve injury-induced neuropathic pain is attenuated in toll-like receptor 2 (TLR2) knock-out mice. In these mice, inflammatory gene expression and spinal cord microglia actvation is compromised, whereas the effects in the dorsal root ganglia (DRG) have not been tested. In this study, we investigated the role of TLR2 in inflammatory responses in the DRG after peripheral nerve injury.ResultsL5 spinal nerve transection injury induced the expression of macrophage-attracting chemokines such as CCL2/MCP-1 and CCL3/MIP-1 and subsequent macrophage infiltration in the DRG of wild-type mice. In TLR2 knock-out mice, however, the induction of chemokine expression and macrophage infiltration following nerve injury were markedly reduced. Similarly, the induction of IL-1β and TNF-α expression in the DRG by spinal nerve injury was ameliorated in TLR2 knock-out mice. The reduced inflammatory response in the DRG was accompanied by attenuation of nerve injury-induced spontaneous pain hypersensitivity in TLR2 knock-out mice.ConclusionsOur data show that TLR2 contributes to nerve injury-induced proinflammatory chemokine/cytokine gene expression and macrophage infiltration in the DRG, which may have relevance in the reduced pain hypersensitivity in TLR2 knock-out mice after spinal nerve injury.

Spinal cord microglia contribute to nerve injury‐induced neuropathic pain. We have previously demonstrated that toll‐like receptor 2 (TLR2) signaling is critical for nerve injury‐induced activation of spinal cord microglia, but the responsible endogenous TLR2 agonist has not been identified. Here, we show that nerve injury‐induced upregulation of sialyltransferase St3gal2 in sensory neurons leads to an increase in expression of the sialylated glycosphingolipid, GT1b. GT1b ganglioside is axonally transported to the spinal cord dorsal horn and contributes to characteristics of neuropathic pain such as mechanical and thermal hypersensitivity. Spinal cord GT1b functions as an TLR2 agonist and induces proinflammatory microglia activation and central sensitization. Pharmacological inhibition of GT1b synthesis attenuates nerve injury‐induced spinal cord microglia activation and pain hypersensitivity. Thus, the St3gal2‐GT1b‐TLR2 axis may offer a novel therapeutic target for the treatment of neuropathic pain.