Melatonin Improves Mitochondrial Dysfunction and Attenuates Neuropathic Pain by Regulating SIRT1 in Dorsal Root Ganglions.

Neuropathic pain is a debilitating chronic pain condition and is refractory to the currently available treatments. Emerging evidence suggests that melatonin exerts analgesic effects in rodent models of neuropathic pain. Nevertheless, the exact underlying mechanisms of the analgesic effects of melatonin on neuropathic pain are largely unknown. Here, we observed that spinal nerve ligation (SNL) in rats L5 and L6 induced an obvious decrease in the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), indicating the induction of mechanical allodynia and the hyperalgesia, and melatonin prevented the genesis and maintenance of mechanical allodynia and the hyperalgesia. Notably, the inhibitory action of melatonin on SNL-induced mechanical allodynia and heat hypersensitivity was inhibited by a SIRT1 inhibitor (EX527). Melatonin treatment increased the expression of neuronal sirtuin1 (SIRT1) in DRGs following nerve injury. Furthermore, melatonin treatment restored the injury-dependent decrease in mitochondrial membrane potential and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and reduced the injury-dependent increase in hydrogen peroxide and 8-hydroxy-2-deoxyguanosine (8-OHdG), which was inhibited by EX527. In addition, we found that EX527 impeded the inhibitory effects of melatonin on the SNL-induced increased expression of cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß). In conclusion, the above data demonstrated that melatonin alleviated mechanical allodynia and hyperalgesia induced by peripheral nerve injury via SIRT1 activation. Melatonin resolved mitochondrial dysfunction-oxidative stress-dependent and neuroinflammation mechanisms that were driven by SIRT1 after nerve injury.

Comparative Effectiveness of Midazolam, Propofol, and Dexmedetomidine in Patients With or at Risk for Acute Respiratory Distress Syndrome: A Propensity Score-Matched Cohort Study.

Background: Sedatives are commonly used in patients with or at risk for acute respiratory distress syndrome (ARDS) during mechanical ventilation. To systematically compare the outcomes of sedation with midazolam, propofol, and dexmedetomidine in patients with or at risk for ARDS. Methods: We developed a dataset of real-world data to enable the comparison of the effectiveness and safety of sedatives and the associated outcomes from the MIMIC-III database and the eICU Collaborative Research database. We performed a systematic study with six cohorts to estimate the relative risks of outcomes among patients administered different sedatives. Propensity score matching was performed to generate a balanced 1:1 matched cohort and to identify potential prognostic factors. The outcomes included hospital mortality, duration of mechanical ventilation, length of intensive care unit stay, length of hospitalization, and likelihood of being discharged home. Results: We performed 60 calibrated analyses among all groups and outcomes with 17,410 eligible patients. Sedation with dexmedetomidine was associated with a lower in-hospital mortality rate than sedation with midazolam and propofol or sedation without dexmedetomidine (p < 0.001). When compared with no sedation, the use of midazolam, propofol or dexmedetomidine was associated with a longer ICU stay and longer hospitalization duration (p < 0.01). Patients treated with midazolam were relatively less likely to be discharged home (p < 0.05). Conclusion: Patients treated with dexmedetomidine had a reduced risk of mortality. These data suggest that dexmedetomidine may be the preferred sedative in patients with or at risk for ARDS.

Foxo1 selectively regulates static mechanical pain by interacting with Nav1.7.

ABSTRACT: Mechanical allodynia is a debilitating condition for millions of patients with chronic pain. Mechanical allodynia can manifest in distinct forms, including brush-evoked dynamic and filament-evoked static allodynia. In the nervous system, the forkhead protein Foxo1 plays a critical role in neuronal structures and functions. However, the role of Foxo1 in the somatosensory signal remains unclear. Here, we found that Foxo1 selectively regulated static mechanical pain. Foxo1 knockdown decreased sensitivity to static mechanical stimuli in normal rats and attenuated static mechanical allodynia in rat models for neuropathic, inflammatory, and chemotherapy pain. Conversely, Foxo1 overexpression selectively enhanced sensitivity to static mechanical stimuli and provoked static mechanical allodynia. Furthermore, Foxo1 interacted with voltage-gated sodium Nav1.7 channels and increased the Nav1.7 current density by accelerating activation rather than by changing the expression of Nav1.7 in dorsal root ganglia neurons. In addition, the serum level of Foxo1 was found to be increased in chronic pain patients and to be positively correlated with the severity of chronic pain. Altogether, our findings suggest that serum Foxo1 level could be used as a biological marker for prediction and diagnosis of chronic pain. Moreover, selective blockade of Foxo1/Nav1.7 interaction may offer a new therapeutic approach in patients with mechanical pain.

Nicotinamide adenine dinucleotide phosphate oxidase 2-derived reactive oxygen species contribute to long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn and persistent mirror-image pain following high-frequency stimulus of the sciatic nerve.

High-frequency stimulation (HFS) of the sciatic nerve has been reported to produce long-term potentiation (LTP) and long-lasting pain hypersensitivity in rats. However, the central underlying mechanism remains unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) belongs to a group of electron-transporting transmembrane enzymes that produce reactive oxygen species (ROS). Here, we found that NOX2 was upregulated in the lumbar spinal dorsal horn after HFS of the left sciatic nerve, which induced bilateral pain and spinal LTP in both male and female rats. Blocking NOX2 with blocking peptide or shRNA prevented the development of bilateral mechanical allodynia, the induction of spinal LTP, and the phosphorylation of N-methyl-d-aspartate (NMDA) receptor 2B (GluN2B) and nuclear factor kappa-B (NF-κB) p65 after HFS. Moreover, NOX2 shRNA reduced the frequency and amplitude of both spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents in laminar II neurons. Furthermore, 8-hydroxyguanine (8-OHG), an oxidative stress marker, was increased in the spinal dorsal horn. Spinal application of ROS scavenger, Phenyl-N-tert-butylnitrone (PBN), depressed the already established spinal LTP. Spinal application of H2O2, one ROS, induced LTP and bilateral mechanical allodynia, increased the frequency and amplitude of spontaneous excitatory postsynaptic currents in laminar II neurons, and phosphorylated GluN2B and p65 in the dorsal horn. This study provided electrophysiological and behavioral evidence that NOX2-derived ROS in the spinal cord contributed to persistent mirror-image pain by enhancing the synaptic transmission, which was mediated by increasing presynaptic glutamate release and activation of NMDA receptor and NF-κB in the spinal dorsal horn.

Activation of liver x receptors prevents the spinal LTP induced by skin/muscle retraction in the thigh via SIRT1/NF-Κb pathway.

It has been reported that skin/muscle incision and retraction (SMIR) in the thigh, produces mechanical allodynia in the hind paw, far from the site of incision/retraction. The mechanical allodynia lasts about 22 days, indicating chronic post-operative pain develops. The precise mechanisms, however, are largely unclear. In the current study, we further found that SMIR surgery induced LTP of c-fiber evoked field potentials that lasted at least 4 h. The mRNA and protein level of tumor necrosis factor-alpha (TNFα) and acetylated nuclear factor-kappaB p65 (ac-NF-κB p65) in the lumbar spinal dorsal horn was gradually increased during LTP development, while pretreatment with either TNFα neutralization antibody or NF-κB inhibitor PDTC completely prevented the induction of LTP. Moreover, the expression of Silent information regulator 1 (SIRT1) in the lumbar spinal dorsal horn was decreased and activation of SIRT1 by SRT1720 also prevented the induction of LTP. Importantly, the spinal expression of Liver X receptors (LXRs) was increased, both at mRNA and protein level following SMIR. Application of LXRs agonist T0901317 to the spinal dorsal horn prevented LTP induction following SMIR. Mechanistically, T0901317 enhanced the expression of SIRT1 and decreased the expression of ac-NF-κB p65 and TNFα. Spinal application of SIRT1 antagonist EX-527, 30 min before T0901317 administration, completely blocked the inhibiting effect of T0901317 on LTP, and on expression of ac-NF-κB p65 and TNFα. These results indicated that activation of LXRs prevented SMIR-induced LTP by inhibiting NF-κB/TNFα pathway via increasing SIRT1 expression.

Rostral ventromedial medulla-mediated descending facilitation following P2X7 receptor activation is involved in the development of chronic post-operative pain.

Chronic postsurgical pain (CPSP) remains a medical problem. Whether the descending modulation of nociceptive transmission from the rostral ventromedial medulla (RVM) plays a role in CPSP induced by skin/muscle incision and retraction (SMIR) in the thigh is still unknown. In this study, we found that SMIR surgery, which induced either bilateral or unilateral mechanical allodynia, activated microglia, and up-regulated interleukin-1ß (IL-1ß), an important cytokine, and 8-hydroxyguanine, an oxidative stress marker in the RVM. In addition, the release of 5-hydroxytryptamine (5-HT) was increased in the ipsilateral and contralateral RVM in rats with either bilateral or unilateral pain following SMIR. The 5-HT level increase, 5-HT3 receptor (5-HT3R) up-regulation, and microglia activation were found bilaterally in SMIR rats with bilateral pain, but only ipsilaterally in SMIR rats with unilateral pain. The intrathecal injection of the 5-HT3R antagonist Y25130 prevented the development of CPSP and the activation of spinal microglia induced by SMIR. Furthermore, P2X7 receptor (P2X7R) was up-regulated in microglia in the RVM. The microinjection of the P2X7R antagonist brilliant blue G (BBG, a non-competitive P2X7R antagonist) into the RVM prevented the development of mechanical allodynia, inhibited the activation of microglia, and decreased the expression of IL-1ß and 8-hydroxyguanine in the RVM following SMIR. Importantly, BBG injected into the RVM also decreased the activation of microglia and the level of 5-HT in the lumbar 3 (L3) spinal cord. The microinjection of the P2X7R agonist BzATP, the NADPH oxidase activator phorbol-12-myristate-13-acetate, or IL-1ß into the RVM induced bilateral mechanical allodynia, microglia activation, and 5-HT release in the L3 spinal dorsal horn. Taken together, P2X7R activation in microglia in the RVM following SMIR might be responsible for the development of CPSP via activating descending serotonergic pathway. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Bulleyaconitine A preferably reduces tetrodotoxin-sensitive sodium current in uninjured dorsal root ganglion neurons of neuropathic rats probably via inhibition of protein kinase C.

Oral Bulleyaconitine A (BLA) is effective for treating neuropathic pain in human patients, but the underlying mechanism is poorly understood. Here, we tested whether BLA blocked voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons. Compelling evidence shows that voltage-gated sodium channels are upregulated in uninjured DRG neurons but downregulated in injured ones following peripheral nerve injury. We found that BLA preferably inhibited Na currents in uninjured DRG neurons in neuropathic rats. Compared to sham rats, IC50 values for resting and inactivated Na currents were 113 and 74 times lower in injured and uninjured neurons of L4-6 DRGs in spared nerve injury (SNI) rats (4.55 and 0.56 nM) and were 688 and 518 times lower in the uninjured L4 and L6 DRG neurons of L5 spinal nerve ligation (L5-SNL) rats. The use-dependent blockage of BLA on Na currents was more potent in neuropathic rats compared to sham rats. Bulleyaconitine A facilitated the inactivation of Na channels in each group. IC50 values for resting and inactivated tetrodotoxin-sensitive (TTX-S) channels were 1855 and 1843 times lower than those for TTX-resistant channels in the uninjured neurons of L5 spinal nerve ligation rats. The upregulation of protein kinase C was associated with the preferable effect of BLA on TTX-S Na channels in the uninjured DRG neurons. Local application of BLA onto L4-6 DRGs at 0.1 to 10 nM dose-dependently alleviated the mechanical allodynia and thermal hyperalgesia in L5 spinal nerve ligation model. Thus, preferable blockage of TTX-S Na channels in uninjured DRG neurons may contribute to BLA's antineuropathic pain effect.

Src-family kinases activation in spinal microglia contributes to central sensitization and chronic pain after lumbar disc herniation.

Background Lumbar disc herniation is a major cause of radicular pain, but the underlying mechanisms remain largely unknown. Spinal activation of src-family kinases are involved in the development of chronic pain from nerve injury, inflammation, and cancer. In the present study, the role of src-family kinases activation in lumbar disc herniation-induced radicular pain was investigated. Results Lumbar disc herniation was induced by implantation of autologous nucleus pulposus, harvest from tail, in lumbar 4/5 spinal nerve roots of rat. Behavior test and electrophysiologic data showed that nucleus pulposus implantation induced persistent mechanical allodynia and thermal hyperalgesia and increased efficiency of synaptic transmission in spinal dorsal horn which underlies central sensitization of pain sensation. Western blotting and immunohistochemistry staining revealed that the expression of phosphorylated src-family kinases was upregulated mainly in spinal microglia of rats with nucleus pulposus. Intrathecal delivery of src-family kinases inhibitor PP2 alleviated pain behaviors, decreased efficiency of spinal synaptic transmission, and reduced phosphorylated src-family kinases expression. Furthermore, we found that the expression of ionized calcium-binding adapter molecule 1 (marker of microglia), tumor necrosis factor-α, interleukin 1 -ß in spinal dorsal horn was increased in rats with nucleus pulposus. Therapeutic effect of PP2 may be related to its capacity in reducing the expression of these factors. Conclusions These findings suggested that central sensitization was involved in radicular pain from lumbar disc herniation; src-family kinases-mediated inflammatory response may be responsible for central sensitization and chronic pain after lumbar disc herniation.

Liver X Receptor α Is Involved in Counteracting Mechanical Allodynia by Inhibiting Neuroinflammation in the Spinal Dorsal Horn.

BACKGROUND: Liver X receptors, including α and ß isoforms, are ligand-activated transcription factors. Whether liver X receptor α plays a role in neuropathic pain is unknown. METHODS: A spared nerve injury model was established in adult male rats and mice. Von Frey tests were performed to evaluate the neuropathic pain behavior; Western blot and immunohistochemistry were performed to understand the underlying mechanisms. RESULTS: Intrathecal injection of a specific liver X receptor agonist T0901317 or GW3965 could either prevent the development of mechanical allodynia or alleviate the established mechanical allodynia, both in rats and wild-type mice. GW3965 could inhibit the activation of glial cells and the expression of tumor necrosis factor-α (mean ± SD: 196 ± 48 vs. 119 ± 57; n = 6; P < 0.01) and interleukin 1ß (mean ± SD: 215 ± 69 vs. 158 ± 74; n = 6; P < 0.01) and increase the expression of interleukin 10 in the spinal dorsal horn. All of the above effects of GW3965 could be abolished by liver X receptor α mutation. Moreover, more glial cells were activated, and more interleukin 1ß was released in the spinal dorsal horn in liver X receptor α knockout mice than in wild-type mice after spared nerve injury. Aminoglutethimide, a neurosteroid synthesis inhibitor, blocked the inhibitory effect of T0901317 on mechanical allodynia, on the activation of glial cells, and on the expression of cytokines. CONCLUSIONS: Activation of liver X receptor α inhibits mechanical allodynia by inhibiting the activation of glial cells and rebalancing cytokines in the spinal dorsal horn via neurosteroids.

EXPRESS: Methylcobalamin ameliorates neuropathic pain induced by vincristine in rats: Effect on loss of peripheral nerve fibers and imbalance of cytokines in the spinal dorsal horn.

BACKGROUND: Vincristine, a widely used chemotherapeutic agent, often induces painful peripheral neuropathy and there are currently no effective drugs to prevent or treat this side effect. Previous studies have shown that methylcobalamin has potential analgesic effect in diabetic and chronic compression of dorsal root ganglion model; however, whether methylcobalamin has effect on vincristine-induced painful peripheral neuropathy is still unknown. RESULTS: We found that vincristine-induced mechanical allodynia and thermal hyperalgesia, accompanied by a significant loss of intraepidermal nerve fibers in the plantar hind paw skin and an increase in the incidence of atypical mitochondria in the sciatic nerve. Moreover, in the spinal dorsal horn, the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the protein expression of p-p65 as well as tumor necrosis factor a was increased, whereas the protein expression of IL-10 was decreased following vincristine treatment. Furthermore, intraperitoneal injection of methylcobalamin could dose dependently attenuate vincristine-induced mechanical allodynia and thermal hyperalgesia, which was associated with intraepidermal nerve fibers rescue, and atypical mitochondria prevalence decrease in the sciatic nerve. Moreover, methylcobalamin inhibited the activation of NADPH oxidase and the downstream NF-kB pathway. Production of tumor necrosis factor a was also decreased and production of IL-10 was increased in the spinal dorsal horn following methylcobalamin treatment. Intrathecal injection of Phorbol-12-Myristate-13-Acetate, a NADPH oxidase activator, could completely block the analgesic effect of methylcobalamin. CONCLUSIONS: Methylcobalamin attenuated vincrinstine-induced neuropathic pain, which was accompanied by inhibition of intraepidermal nerve fibers loss and mitochondria impairment. Inhibiting the activation of NADPH oxidase and the downstream NF-kB pathway, resulting in the rebalancing of proinflammatory and anti-inflammatory cytokines in the spinal dorsal horn might also be involved. These findings might provide potential target for preventing vincristine-induced neuropathic pain.