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This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Lappaconitine is a representative C18-diterpenoid alkaloid extracted from Aconitum sinomontanum Nakai and has been prescribed as a pain relief medicine in China for more than 30 years. This study evaluated its antihypersensitivity activity in the rat models of neuropathic and cancer pains and explored its underlying mechanisms. Subcutaneous injection of cumulative doses of lappaconitine produced dose-dependent mechanical antiallodynia and thermal antihyperalgesia in spinal nerve ligation-induced neuropathic rats. The cumulative dose-response analysis exhibited their E max values of 53.3 and 58.3% MPE, and ED 50 values of 1.1 and 1.6 mg/kg. Single intrathecal lappaconitine dose in neuropathy also dose- and time-dependently blocked mechanical allodynia, with an E max of 66.1% MPE and an ED 50 of 0.8 μg. Its multiple twice-daily intrathecal administration over 7 days did not induce mechanical antiallodynic tolerance. Subcutaneous cumulative doses of lappaconitine also produced dose-dependent blockade of mechanical allodynia in the rat bone cancer pain model induced by tibia implantation of cancer cells, with the E max of 57.9% MPE and ED 50 of 2.0 mg/kg. Furthermore, lappaconitine treatment stimulated spinal dynorphin A expression in neuropathic rats, and in primary cultures of microglia but not neurons or astrocytes. Intrathecal pretreatment with the specific microglia depletor liposome-encapsulated clodronate, dynorphin A antibody, and κ-opioid receptor antagonist GNTI totally suppressed intrathecal and subcutaneous lappaconitine-induced mechanical antiallodynia. This study suggests that lappaconitine exhibits antinociception through directly stimulating spinal microglial dynorphin A expression. Graphical Abstract ᅟ.

Background: Prolonged morphine treatment leads to antinociceptive tolerance. Suppression of spinal astrocytes or d-amino acid oxidase (DAAO), an astroglial enzyme catalyzing oxidation of d-amino acids, has reversed morphine antinociceptive tolerance. Since the astrocyte-DAAO pathway generates hydrogen peroxide, an agonist of the TRPA1 channel expressed spinally on nociceptive nerve terminals and astrocytes, we tested a hypothesis that the spinal TRPA1 contributes to antinociceptive tolerance to prolonged spinal morphine treatment., Methods: Nociception was assessed using hot-plate test in rats with an intrathecal (it) catheter. Drugs were administered it twice daily from day one to seven in five treatment groups: (i) Saline, (ii) Chembridge-5861528 (a TRPA1 antagonist; 10μg), (iii) morphine (10μg), (iv) Chembridge-5861528 (10μg)+morphine (10μg), (v) DMSO. Antinociceptive action of morphine was assessed at day one and eight. Additionally, mRNA for DAAO and TRPA1 in the spinal cord was determined on day 8., Results: Morphine treatment produced antinociceptive tolerance, which was attenuated by co-administration of Chembridge-5861528 that alone had no effect on hot-plate latencies. In animals treated with morphine only, spinal mRNA for DAAO but not TRPA1 was increased. DAAO increase was prevented by co-administration of Chembridge-5861528., Conclusions: Antinociceptive morphine tolerance and up-regulation of spinal DAAO were attenuated in morphine-treated animals by blocking the spinal TRPA1. This finding suggests that spinal TRPA1 may contribute, at least partly, to facilitation of morphine antinociceptive tolerance through mechanisms that possibly involve TRPA1-mediated up-regulation of the astroglial DAAO, a generator of hydrogen peroxide, a pronociceptive compound acting also on TRPA1., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Background: D-Amino acid oxidase (DAAO) is a flavin adenine dinucleotide-dependent peroxisomal flavoenzyme which is almost exclusively expressed within astrocytes in the spinal cord. DAAO catalyzes oxidation of D-amino acids to hydrogen peroxide, which is a stable and less active reactive oxygen species, and may represent a final form of reactive oxygen species. This study tested the hypothesis that the spinal astroglial DAAO-hydrogen peroxide pathway plays an important role in the development of morphine antinociceptive tolerance., Methods: Rat and mouse formalin, hot-plate, and tail-flick tests were used, and spinal DAAO expression and hydrogen peroxide level were measured. Sample size of animals was six in each study group., Results: Subcutaneous and intrathecal DAAO inhibitors, including 5-chloro-benzo[d]isoxazol-3-ol, AS057278, and sodium benzoate, completely prevented and reversed morphine antinociceptive tolerance in the formalin, hot-plate, and tail-immersion tests, with a positive correlation to their DAAO inhibitory activities. Intrathecal gene silencers, small interfering RNA/DAAO and small hairpin RNA/DAAO, almost completely prevented morphine tolerance. Intrathecal 5-chloro-benzo[d]isoxazol-3-ol and small interfering RNA/DAAO completely prevented increased spinal hydrogen peroxide levels after chronic morphine treatment. Intrathecal nonselective hydrogen peroxide scavenger phenyl-tert-N-butyl nitrone and the specific hydrogen peroxide catalyst catalase also abolished established morphine tolerance. Spinal dorsal horn astrocytes specifically expressed DAAO was significantly up-regulated, accompanying astrocyte hypertrophy after chronic morphine treatment., Conclusions: For the first time, the authors' result identify a novel spinal astroglial DAAO-hydrogen peroxide pathway that is critically involved in the initiation and maintenance of morphine antinociceptive tolerance, and suggest that this pathway is of potential utility for the management of morphine tolerance and chronic pain.

We studied the hypothesis that spinal d-amino acid oxidase (DAAO) that is expressed in astrocytes and that has been reported to promote tonic pain in various pathophysiological conditions plays a role in 'physiological' pain hypersensitivity induced by rapid eye movement sleep deprivation (REMSD). The experiments were performed in healthy rats with a chronic intrathecal (i.t.) catheter. Pain behavior was assessed by determining limb withdrawal response to repetitive stimulation of the hind paw with a calibrated series of monofilaments. REMSD of 48 h duration produced a significant mechanical hypersensitivity. At 48 h of REMSD, the animals were treated i.t. with a DAAO inhibitor or vehicle. Three structurally different DAAO inhibitors were tested in this study: 6-chlorobenzo[d]isoxazol-3-ol (CBIO), sodium benzoate, and 5-methylpyrazole-3-carboxylic acid (AS-057278). CBIO (1-3 μg), sodium benzoate (30-100 μg) and AS-057278 (3-10 μg) produced dose-related antihypersensitivity effects in sleep-deprived animals. In control animals (with no sleep deprivation), the currently used doses of DAAO inhibitors failed to produce significant changes in mechanically evoked pain behavior. The results indicate that among spinal pain facilitatory mechanisms that contribute to the sleep deprivation-induced mechanical pain hypersensitivity is DAAO, presumably due to production of reactive oxygen species, such as hydrogen peroxide, an endogenous agonist of the pronociceptive TRPA1 ion channel., (© 2013.)

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spinal d -amino acid oxidase (DAAO) is an FAD-dependent peroxisomal flavoenzyme which mediates the conversion of neutral and polar d -amino acids (including d -serine) to the corresponding α-keto acids, and simultaneously produces hydrogen peroxide and ammonia. This study has aimed to explore the potential contributions of spinal DAAO and its mediated hydrogen peroxide/ d -serine metabolism to the development of morphine-induced hyperalgesia. Bi-daily subcutaneous injections of morphine to mice over 7 days induced thermal hyperalgesia as measured by both the hot-plate and tail-immersion tests, and spinal astroglial activation with increased spinal gene expression of DAAO, glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Subcutaneous injections of the potent DAAO inhibitor CBIO (5-chloro-benzo[ d ]isoxazol-3-ol) prevented and reversed the chronic morphine-induced hyperalgesia. CBIO also inhibited both astrocyte activation and the expression of pro-inflammatory cytokines. Intrathecal injection of the hydrogen peroxide scavenger PBN (phenyl- N - tert -butylnitrone) and of catalase completely reversed established morphine hyperalgesia, whereas subcutaneous injections of exogenous d -serine failed to alter chronic morphine-induced hyperalgesia. These results provided evidence that spinal DAAO and its subsequent production of hydrogen peroxide rather than the d -serine metabolism contributed to the development of morphine-induced hyperalgesia. [ABSTRACT FROM AUTHOR]

• Gabapentinoids are considered first-line medications for neuropathic pain. • Intrathecal gabapentinoids produced rapid antihypersensitivity in neuropathic rats. • Spinal microglial IL-10 and β-endorphin mediated gabapentinoids antiallodynia. Gabapentinoids are recommended first-line treatments for neuropathic pain. They are neuronal voltage-dependent calcium channel α2δ-1 subunit ligands and have been suggested to attenuate neuropathic pain via interaction with neuronal α2δ-1 subunit. However, the current study revealed their microglial mechanisms underlying antineuropathic pain. Intrathecal injection of gabapentin, pregabalin and mirogabalin rapidly inhibited mechanical allodynia and thermal hyperalgesia, with projected ED 50 values of 30.3, 6.2 and 1.5 µg (or 176.9, 38.9 and 7.2 nmol) and E max values of 66%, 61% and 65% MPE respectively for mechanical allodynia. Intrathecal gabapentinoids stimulated spinal mRNA and protein expression of IL-10 and β-endorphin (but not dynorphin A) in neuropathic rats with the time point parallel to their inhibition of allodynia, which was observed in microglia but not astrocytes or neurons in spinal dorsal horns by using double immunofluorescence staining. Intrathecal gabapentin alleviated pain hypersensitivity in male/female neuropathic but not male sham rats, whereas it increased expression of spinal IL-10 and β-endorphin in male/female neuropathic and male sham rats. Treatment with gabapentin, pregabalin and mirogabalin specifically upregulated IL-10 and β-endorphin mRNA and protein expression in primary spinal microglial but not astrocytic or neuronal cells, with EC 50 values of 41.3, 11.5 and 2.5 µM and 34.7, 13.3 and 2.8 µM respectively. Pretreatment with intrathecal microglial metabolic inhibitor minocycline, IL-10 antibody, β-endorphin antiserum or μ-opioid receptor antagonist CTAP (but not κ- or δ-opioid receptor antagonists) suppressed spinal gabapentinoids-inhibited mechanical allodynia. Immunofluorescence staining exhibited specific α2δ-1 expression in neurons but not microglia or astrocytes in the spinal dorsal horns or cultured primary spinal cells. Thus the results illustrate that gabapentinoids alleviate neuropathic pain through stimulating expression of spinal microglial IL-10 and consequent β-endorphin. [ABSTRACT FROM AUTHOR]

[Display omitted] Thalidomide is an antiinflammatory, antiangiogenic and immunomodulatory agent which has been used for the treatment of erythema nodosum leprosum and multiple myeloma. It has also been employed in treating complex regional pain syndromes. The current study aimed to reveal the molecular mechanisms underlying thalidomide-induced pain antihypersensitive effects in neuropathic pain. Thalidomide gavage, but not its more potent analogs lenalidomide and pomalidomide, inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain rats induced by tight ligation of spinal nerves, with ED 50 values of 44.9 and 23.5 mg/kg, and E max values of 74% and 84% MPE respectively. Intrathecal injection of thalidomide also inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain. Treatment with thalidomide, lenalidomide and pomalidomide reduced peripheral nerve injury-induced proinflammatory cytokines (TNFα, IL-1β and IL-6) in the ipsilateral spinal cords of neuropathic rats and LPS-treated primary microglial cells. In contrast, treatment with thalidomide, but not lenalidomide or pomalidomide, stimulated spinal expressions of IL-10 and β-endorphin in neuropathic rats. Particularly, thalidomide specifically stimulated IL-10 and β-endorphin expressions in microglia but not astrocytes or neurons. Furthermore, pretreatment with the IL-10 antibody blocked upregulation of β-endorphin in neuropathic rats and cultured microglial cells, whereas it did not restore thalidomide-induced downregulation of proinflammatory cytokine expression. Importantly, pretreatment with intrathecal injection of the microglial metabolic inhibitor minocycline, IL-10 antibody, β-endorphin antiserum, and preferred or selective μ-opioid receptor antagonist naloxone or CTAP entirely blocked thalidomide gavage-induced mechanical antiallodynia. Our results demonstrate that thalidomide, but not lenalidomide or pomalidomide, alleviates neuropathic pain, which is mediated by upregulation of spinal microglial IL-10/β-endorphin expression, rather than downregulation of TNFα expression. [ABSTRACT FROM AUTHOR]