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-1β (IL-1β). 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-Noxide 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.

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, Toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the non-opioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knockouts of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4 −/− mice had reduced oxycodone-induced p38 and JNK phosphorylation, whilst displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system which amplifies opioid-induced elevations in extracellular dopamine levels and therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Exercise is known to exert a systemic anti-inflammatory influence, but whether its effects are sufficient to protect against subsequent neuropathic pain is underinvestigated. We report that 6 weeks of voluntary wheel running terminating before chronic constriction injury (CCI) prevented the full development of allodynia for the ∼3-month duration of the injury. Neuroimmune signaling was assessed at 3 and 14 days after CCI. Prior exercise normalized ipsilateral dorsal spinal cord expression of neuroexcitatory interleukin (IL)-1β production and the attendant glutamate transporter GLT-1 decrease, as well as expression of the disinhibitory P2X4R-BDNF axis. The expression of the macrophage marker Iba1 and the chemokine CCL2 (MCP-1), and a neuronal injury marker (activating transcription factor 3), was attenuated by prior running in the ipsilateral lumbar dorsal root ganglia. Prior exercise suppressed macrophage infiltration and/or injury site proliferation, given decreased presence of macrophage markers Iba1, iNOS (M1), and Arg-1 (M2; expression was time dependent). Chronic constriction injury–driven increases in serum proinflammatory chemokines were suppressed by prior running, whereas IL-10 was increased. Peripheral blood mononuclear cells were also stimulated with lipopolysaccharide ex vivo, wherein CCI-induced increases in IL-1β, nitrite, and IL-10 were suppressed by prior exercise. Last, unrestricted voluntary wheel running, beginning either the day of, or 2 weeks after, CCI, progressively reversed neuropathic pain. This study is the first to investigate the behavioral and neuroimmune consequences of regular exercise terminating before nerve injury. This study suggests that chronic pain should be considered a component of “the diseasome of physical inactivity,” and that an active lifestyle may prevent neuropathic pain.

SUMMARY 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-1β 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-1β mRNA was attenuated by E2 in female but not male adult hippocampal microglia. However, females supplemented with E2 in vivo produced a potentiated IL-1β 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.

The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a G- and G-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naïve male rats intrathecally transfected with G (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO), which was abolished by intrathecal interleukin-1 receptor antagonist. Chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in male rats intrathecally transfected with G (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with G or G DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing G-DREADDs, and inhibited lipopolysaccharide- and CCL2-induced inflammatory signaling from cells expressing G-DREADDs. These studies are the first to manipulate microglia function using DREADDs, which allow the role of glia in pain to be conclusively demonstrated, unconfounded by neuronal off-target effects that exist for all other drugs that also inhibit glia. Hence, these studies are the first to conclusively demonstrate that in vivo stimulation of resident spinal microglia in intact spinal cord is a) sufficient for allodynia, and b) necessary for allodynia induced by peripheral nerve injury. DREADDs are a unique tool to selectively explore the physiological and pathological role of microglia in vivo.

We have recently reported that a short course of morphine, starting 10days after sciatic chronic constriction injury (CCI), prolonged the duration of mechanical allodynia for months after morphine ceased. Maintenance of this morphine-induced persistent sensitization was dependent on spinal NOD-like receptor protein 3 (NLRP3) inflammasomes-protein complexes that proteolytically activate interleukin-1β (IL-1β) via caspase-1. However, it is still unclear how NLRP3 inflammasome signaling is maintained long after morphine is cleared. Here, we demonstrate that spinal levels of the damage associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1) and biglycan are elevated during morphine-induced persistent sensitization in male rats; that is, 5weeks after cessation of morphine dosing. We also show that HMGB1 and biglycan levels are at least partly dependent on the initial activation of caspase-1, as well as Toll like receptor 4 (TLR4) and the purinergic receptor P2X7R-receptors responsible for priming and activation of NLRP3 inflammasomes. Finally, pharmacological attenuation of the DAMPs HMGB1, biglycan, heat shock protein 90 and fibronectin persistently reversed morphine-prolonged allodynia. We conclude that after peripheral nerve injury, morphine treatment results in persistent DAMP release via TLR4, P2X7R and caspase-1, which are involved in formation/activation of NLRP3 inflammasomes. These DAMPs are responsible for maintaining persistent allodynia, which may be due to engagement of a positive feedback loop, in which NLRP3 inflammasomes are persistently activated by DAMPs signaling at TLR4 and P2X7R.

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at TLR4, presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu-opioid receptor (MOR) inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated NFκB, increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and prostaglandin E2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequalae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by presumptive glial inhibitors, as well as TLR4-mediated phenomena more broadly.

While stress and stress-induced glucocorticoids are classically considered immunosuppressive, they can also enhance proinflammatory responses to subsequent challenges. Corticosterone (CORT) primes rat immune cells, exacerbating pro-inflammatory responses to subsequent immune challenges. Stress can also sensitize pain. One possibility is that stress primes spinal immune cells, predominantly glia, which are key mediators in pain enhancement through their release of proinflammatory cytokines. Therefore, we aimed to identify whether prior CORT sensitizes spinal cord glia such that a potentiated pro-inflammatory response occurs to later intrathecal (IT) lipopolysaccharide (LPS), thereby enhancing pain. Rats received subcutaneous CORT/vehicle 24 h before IT LPS/vehicle. Hind paw pain thresholds were measured before CORT/vehicle, before and up to 48 h after IT LPS/vehicle. In separate rats treated as above, lumbar spinal cord tissue was collected and processed for proinflammatory mediators. CORT alone had no effect on pain responses, nor on any pro-inflammatory cytokines measured. LPS induced allodynia (decreased pain threshold) lasting <4 h and elevated spinal IL-1β and IL-6 protein. Prior CORT potentiated allodynia, lasting >24 h following LPS and potentiated spinal IL-1 and IL-6 protein. Coadministration of IL-1 receptor antagonist with LPS IT completely blocked the allodynia irrespective of whether the system was primed by CORT or not. At 24 h, TLR2, TLR4, MD2 and CD14 mRNAs were significantly elevated within the spinal cord in the CORT+LPS group compared to all other groups. Prior CORT before a direct spinal immune challenge is able to potentiate pain responses and pro-inflammatory cytokine production.