Repeated Morphine Prolongs Postoperative Pain in Male Rats.

BACKGROUND: Opioids are effective postoperative analgesics. Disturbingly, we have previously reported that opioids such as morphine can worsen inflammatory pain and peripheral and central neuropathic pain. These deleterious effects are mediated by immune mediators that promote neuronal hyperexcitability in the spinal dorsal horn. Herein, we tested whether perioperative morphine could similarly prolong postoperative pain in male rats. METHODS: Rats were treated with morphine for 7 days, beginning immediately after laparotomy, while the morphine was tapered in a second group. Expression of genes for inflammatory mediators was quantified in the spinal dorsal horn. In the final experiment, morphine was administered before laparotomy for 7 days. RESULTS: We found that morphine treatment after laparotomy extended postoperative pain by more than 3 weeks (time × treatment: P < .001; time: P < .001; treatment: P < .05). Extension of postoperative pain was not related to morphine withdrawal, as it was not prevented by dose tapering (time × treatment: P = .8; time: P < .001; treatment: P = .9). Prolonged postsurgical pain was associated with increased expression of inflammatory genes, including those encoding Toll-like receptor 4, NOD like receptor protein 3 (NLRP3), nuclear factor kappa B (NFκB), caspase-1, interleukin-1ß, and tumor necrosis factor (P < .05). Finally, we showed that of preoperative morphine, concluding immediately before laparotomy, similarly prolonged postoperative pain (time × treatment: P < .001; time: P < .001; treatment: P < .001). There is a critical window for morphine potentiation of pain, as a 7-day course of morphine that concluded 1 week before laparotomy did not prolong postsurgical pain. CONCLUSIONS: These studies indicate the morphine can have a deleterious effect on postoperative pain. These studies further suggest that longitudinal studies could be performed to test whether opioids similarly prolong postoperative pain in the clinic.

Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation.

Opioid use for pain management has dramatically increased, with little assessment of potential pathophysiological consequences for the primary pain condition. Here, a short course of morphine, starting 10 d after injury in male rats, paradoxically and remarkably doubled the duration of chronic constriction injury (CCI)-allodynia, months after morphine ceased. No such effect of opioids on neuropathic pain has previously been reported. Using pharmacologic and genetic approaches, we discovered that the initiation and maintenance of this multimonth prolongation of neuropathic pain was mediated by a previously unidentified mechanism for spinal cord and pain-namely, morphine-induced spinal NOD-like receptor protein 3 (NLRP3) inflammasomes and associated release of interleukin-1ß (IL-1ß). As spinal dorsal horn microglia expressed this signaling platform, these cells were selectively inhibited in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Multiday treatment with the DREADD-specific ligand clozapine-N-oxide prevented and enduringly reversed morphine-induced persistent sensitization for weeks to months after cessation of clozapine-N-oxide. These data demonstrate both the critical importance of microglia and that maintenance of chronic pain created by early exposure to opioids can be disrupted, resetting pain to normal. These data also provide strong support for the recent "two-hit hypothesis" of microglial priming, leading to exaggerated reactivity after the second challenge, documented here in the context of nerve injury followed by morphine. This study predicts that prolonged pain is an unrealized and clinically concerning consequence of the abundant use of opioids in chronic pain.

Prior exposure to repeated morphine potentiates mechanical allodynia induced by peripheral inflammation and neuropathy.

Opioids, such as morphine, induce potent analgesia and are the gold standard for the treatment of acute pain. However, opioids also activate glia, inducing pro-inflammatory cytokine and chemokine production, which counter-regulates the analgesic properties of classical opioid receptor activation. It is not known how long these adverse pro-inflammatory effects last or whether prior morphine could sensitize the central nervous system (CNS) such that responses to a subsequent injury/inflammation would be exacerbated. Here, multiple models of inflammation or injury were induced two days after morphine (5mg/kg b.i.d., five days , s.c.) to test the generality of morphine sensitization of later pain. Prior repeated morphine potentiated the duration of allodynia from peripheral inflammatory challenges (complete Freund's adjuvant (CFA) into either hind paw skin or masseter muscle) and from peripheral neuropathy (mild chronic constriction injury (CCI) of the sciatic nerve). Spinal cord and trigeminal nucleus caudalis mRNAs were analyzed to identify whether repeated morphine was sufficient to alter CNS expression of pro-inflammatory response genes, measured two days after cessation of treatment. Prior morphine elevated IL-1ß mRNA at both sites, MHC-II and TLR4 in the trigeminal nucleus caudalis but not spinal cord, but not glial activation markers at either site. Finally, in order to identify whether morphine sensitized pro-inflammatory cytokine release, spinal cord was isolated two days after morphine dosing for five days , and slices stimulated ex vivo with lipopolysaccharide. The morphine significantly induced TNFα protein release. Therefore, repeated morphine is able to sensitize subsequent CNS responses to immune challenges.

Enduring reversal of neuropathic pain by a single intrathecal injection of adenosine 2A receptor agonists: a novel therapy for neuropathic pain.

Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A(2A)Rs) decrease proinflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain and that IL-10 can suppress such pain, we evaluated the effects of intrathecally administered A(2A)R agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single intrathecal injection of the A(2A)R agonists 4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl)piperidine-1-carboxylic acid methyl ester (ATL313) or 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine HCl (CGS21680), 10-14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 weeks. Neither drug altered the nociceptive responses of sham-operated controls. An A(2A)R antagonist [ZM241385 (4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol)] coadministered intrathecally with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia but had no effect on allodynia in the absence of the A(2A)R agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4-L6 spinal cord segments both 1 and 4 weeks after a single intrathecal ATL313 administration. Neutralizing IL-10 antibodies administered intrathecally transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A(2A)Rs after intrathecal administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS.

Reduction of opioid withdrawal and potentiation of acute opioid analgesia by systemic AV411 (ibudilast).

Morphine-induced glial proinflammatory responses have been documented to contribute to tolerance to opioid analgesia. Here, we examined whether drugs previously shown to suppress glial proinflammatory responses can alter other clinically relevant opioid effects; namely, withdrawal or acute analgesia. AV411 (ibudilast) and minocycline, drugs with distinct mechanisms of action that result in attenuation of glial proinflammatory responses, each reduced naloxone-precipitated withdrawal. Analysis of brain nuclei associated with opioid withdrawal revealed that morphine altered expression of glial activation markers, cytokines, chemokines, and a neurotrophic factor. AV411 attenuated many of these morphine-induced effects. AV411 also protected against spontaneous withdrawal-induced hyperactivity and weight loss recorded across a 12-day timecourse. Notably, in the spontaneous withdrawal study, AV411 treatment was delayed relative to the start of the morphine regimen so to also test whether AV411 could still be effective in the face of established morphine dependence, which it was. AV411 did not simply attenuate all opioid effects, as co-administering AV411 with morphine or oxycodone caused three-to-five-fold increases in acute analgesic potency, as revealed by leftward shifts in the analgesic dose response curves. Timecourse analyses revealed that plasma morphine levels were not altered by AV411, suggestive that potentiated analgesia was not simply due to prolongation of morphine exposure or increased plasma concentrations. These data support and extend similar potentiation of acute opioid analgesia by minocycline, again providing converging lines of evidence of glial involvement. Hence, suppression of glial proinflammatory responses can significantly reduce opioid withdrawal, while improving analgesia.

Minocycline suppresses morphine-induced respiratory depression, suppresses morphine-induced reward, and enhances systemic morphine-induced analgesia.

Recent data suggest that opioids can activate immune-like cells of the central nervous system (glia). This opioid-induced glial activation is associated with decreased analgesia, owing to the release of proinflammatory mediators. Here, we examine in rats whether the putative microglial inhibitor, minocycline, may affect morphine-induced respiratory depression and/or morphine-induced reward (conditioned place preference). Systemic co-administration of minocycline significantly attenuated morphine-induced reductions in tidal volume, minute volume, inspiratory force, and expiratory force, but did not affect morphine-induced reductions in respiratory rate. Minocycline attenuation of respiratory depression was also paralleled with significant attenuation by minocycline of morphine-induced reductions in blood oxygen saturation. Minocycline also attenuated morphine conditioned place preference. Minocycline did not simply reduce all actions of morphine, as morphine analgesia was significantly potentiated by minocycline co-administration. Lastly, morphine dose-dependently increased cyclooxygenase-1 gene expression in a rat microglial cell line, an effect that was dose-dependently blocked by minocycline. Together, these data support that morphine can directly activate microglia in a minocycline-suppressible manner and suggest a pivotal role for minocycline-sensitive processes in the mechanisms of morphine-induced respiration depression, reward, and pain modulation.

Sex and estradiol influence glial pro-inflammatory responses to lipopolysaccharide in rats.

There is a greater prevalence of neuroinflammatory diseases in females than males. Microglia, the major immunocompetent cells of the central nervous system, play a key role in neuroinflammation. We aimed to determine if inherent differences in toll-like receptor 4 mediated pro-inflammatory response in glia could possibly contribute to the skewed female prevalence of neuroinflammatory disorders. In addition, in order to identify if estradiol (E2), the major female sex steroid contributes to a heightened pro-inflammatory response, estradiol was added both in vivo and in vitro. Microglia and astrocytes were isolated from neonatal pups and stimulated with lipopolysaccharide (LPS) in the presence and absence of E2. Hippocampal microglia were isolated from adult male and female rats and stimulated ex vivo with LPS. Male neonatal microglia and astrocytes produced greater IL-1ß mRNA than females. However, when co-incubated with varying doses of estradiol (E2), the E2 produced anti-inflammatory effects in the male microglia but a pro-inflammatory effect in female microglia. LPS-induced IL-1ß mRNA was attenuated by E2 in female but not male adult hippocampal microglia. However, females supplemented with E2 in vivo produced a potentiated IL-1ß mRNA response. TLR4 mRNA was decreased by LPS in both microglia and astrocytes but was not affected by sex or E2. CD14 mRNA was increased by LPS and may be elevated more in females than males in microglia but not astrocytes. Therefore, sexual dimorphic differences do occur in both neonatal and adult microglia though maturity of the microglia at the time of isolation influences the pro-inflammatory response.