Accepted

Burn injury-induced pain (BIP) is an extremely complicated condition usually resistant to analgesic drugs, while its pathogenesis remains unknown. Considerable attention has been attracted to elucidate the glial mechanisms in chronic pain. In this study, we initiatively used a mouse model of second-degree BIP to investigate the underlying non-neuronal mechanisms at the spinal cord level. Our behavioral results showed that hind-paw burn injury caused persistent allodynia and hyperalgesia for 2 weeks in mice. Further studies revealed that both microglia and astrocytes activated in a spatially- and temporally-dependent manner in spinal cord after burn injury. In addition, the phosphorylated p38 mitogen-activated protein kinase (MAPK)-mediated tumor necrosis factor (TNF) release in spinal microglia is essentially attributed to the early stage of BIP, while the c-Jun N-terminal kinase (JNK) MAPK-dependent chemokine CXCL1 expression is mainly involved in the maintenance of pain hypersensitivity. Most strikingly, burn injury-induced pain symptoms and the activation of astrocytes were significantly suppressed by TNF inhibitor Thalidomide. On the contrary, intrathecal injection of TNF caused apparent pain hypersensitivity, accompanied by the activation of astrocytes and the upregulation of CXCL1 via the JNK MAPK signaling pathway, indicating that TNF is the key cytokine in the interaction between microglia and astrocytes at the spinal level. Moreover, treatment with the CXCR2 receptor antagonist SB225002 to block the biological activities of CXCL1 significantly attenuated the mechanical allodynia and thermal hyperalgesia in this BIP model. Taken together, this study indicates that intervention of glial pathways provides a new perspective in the management of BIP.

Opioids, and numerous centrally active drugs, are metabolized by cytochrome P450 2D (CYP2D). There are sex and estrous cycle differences in brain oxycodone analgesia. Here we investigated the mechanism examining the selective role of CYP2D in the brain on sex, estrous cycle, and hormonal regulation. Propranolol, CYP2D-specific mechanism-based inhibitor, or vehicle was delivered into cerebral ventricles 24 hours before administering oxycodone (or oxymorphone, negative control) orally to male and female (in estrus and diestrus) rats. Ovariectomized and sham-operated females received no treatment, estradiol, progesterone or vehicle. Analgesia was measured using tail-flick latency, and brain drug and metabolite concentrations were measured by microdialysis. Data were analyzed by two-way or mixed ANOVA. Following propranolol (versus vehicle) inhibition and oral oxycodone, there were greater increases in brain oxycodone concentrations and analgesia, and greater decreases in brain oxymorphone/oxycodone ratios (an in vivo phenotype of CYP2D in brain) in males and females in estrus, compared to females in diestrus; with no impact on plasma drug concentrations. There was no impact of propranolol pre-treatment, sex, or cycle after oral oxymorphone (non-CYP2D substrate) on brain oxymorphone concentrations or analgesia. There was no impact of propranolol pre-treatment following ovariectomy on brain oxycodone concentrations or analgesia, which was restored in ovariectomized females following estradiol, but not progesterone, treatment. Sex, cycle, and estradiol regulation of CYP2D in brain in turn altered brain oxycodone concentration and response, which may contribute to the large inter-individual variation in response to the numerous centrally acting CYP2D substrate drugs, including opioids.

The anti-diabetic drug metformin has been shown to reduce pain hypersensitivity in preclinical models of chronic pain and in neuropathic pain in humans. Multiple intracellular pathways have been described as metformin targets. Among them, metformin is an activator of the adenosine 5'-monophosphate protein kinase (AMPK) that can in turn modulate the activity of the E3 ubiquitin ligase NEDD4-2 and thus posttranslational expression of voltage gated sodium channels (Nas). In this study, we found that the bulk of the effect of metformin on Na1.7 is dependent on NEDD4-2. In HEK cells, the expression of Na1.7 at the membrane fraction, obtained by a biotinylation approach, is only reduced by metformin when co-transfected with NEDD4-2. Similarly, in voltage clamp recordings, metformin significantly reduced Na1.7 current density when co-transfected with NEDD4-2. In mouse dorsal root ganglion (DRG) neurons, without changing the biophysical properties of Na1.7, metformin significantly decreased Na1.7 current densities, but not in Nedd4L knockout mice ( ). In addition, metformin induced a significant reduction in NEDD4-2 phosphorylation at the Serine 328 residue in DRG neurons, an inhibitory phosphorylation site of NEDD4-2. In current clamp recordings, metformin reduced the number of action potentials elicited by DRG neurons from , with a partial decrease also present in mice, suggesting that metformin can also change neuronal excitability in an NEDD4-2-independent manner. We suggest that NEDD4-2 is a player for the effect of metformin on the excitability of nociceptive neurons; this action may contribute to the relief of neuropathic pain.Metformin is a multi-target, anti-diabetic drug that has shown therapeutic potential to reduce neuropathic pain. The intracellular mechanisms leading to a reduction in hyperexcitability and pain hypersensitivity remain unclear. We found that metformin acts through the activity of the E3-ubiquitin ligase NEDD4-2 to reduce cell surface expression and currents of voltage gated sodium channels (Nas), especially the Na1.7 isoform. In current clamp experiments, metformin reduced the DRG neuron firing frequency, with a smaller effect in knockout mice ( ). On the other hand, NEDD4-2 is indispensable for the metformin effect on the rheobase and the resting membrane potential of DRG neurons. These results suggest that NEDD4-2 activity is a crucial actor, although not exclusive, to reduce the excitability of nociceptive neurons.