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Abstract: Opioids have been discovered to have Toll-like receptor (TLR) activity, beyond actions at classical opioid receptors. This raises the question whether other pharmacotherapies for pain control may also possess TLR activity, contributing to or opposing their clinical effects. We document that tricyclics can alter TLR4 and TLR2 signaling. In silico simulations revealed that several tricyclics docked to the same binding pocket on the TLR accessory protein, myeloid differentiation protein 2 (MD-2), as do opioids. Eight tricyclics were tested for effects on TLR4 signaling in HEK293 cells over-expressing human TLR4. Six exhibited mild (desipramine), moderate (mianserin, cyclobenzaprine, imiprimine, ketotifen) or strong (amitriptyline) TLR4 inhibition, and no TLR4 activation. In contrast, carbamazepine and oxcarbazepine exhibited mild and strong TLR4 activation, respectively, and no TLR4 inhibition. Amitriptyline but not carbamazepine also significantly inhibited TLR2 signaling in a comparable cell line. Live imaging of TLR4 activation in RAW264.7 cells and TLR4-dependent interleukin-1 release from BV-2 microglia revealed that amitriptyline blocked TLR4 signaling. Lastly, tricyclics with no (carbamazepine), moderate (cyclobenzeprine), and strong (amitriptyline) TLR4 inhibition were tested intrathecally (rats) and amitriptyline tested systemically in wildtype and knockout mice (TLR4 or MyD88). While tricyclics had no effect on basal pain responsivity, they potentiated morphine analgesia in rank-order with their potency as TLR4 inhibitors. This occurred in a TLR4/MyD88-dependent manner as no potentiation of morphine analgesia by amitriptyline occurred in these knockout mice. This suggests that TLR2 and TLR4 inhibition, possibly by interactions with MD2, contributes to effects of tricyclics in vivo. These studies provide converging lines of evidence that several tricyclics or their active metabolites may exert their biological actions, in part, via modulation of TLR4 and TLR2 signaling and suggest that inhibition of TLR4 and TLR2 signaling may potentially contribute to the efficacy of tricyclics in treating chronic pain and enhancing the analgesic efficacy of opioids. [Copyright &y& Elsevier]

Abstract: Opioid-induced glial activation and its proinflammatory consequences have been associated with both reduced acute opioid analgesia and the enhanced development of tolerance, hyperalgesia and allodynia following chronic opioid administration. Intriguingly, recent evidence demonstrates that these effects can result independently from the activation of classical, stereoselective opioid receptors. Here, a structurally disparate range of opioids cause activation of signaling by the innate immune receptor toll like receptor 4 (TLR4), resulting in proinflammatory glial activation. In the present series of studies, we demonstrate that the (+)-isomers of methadone and morphine, which bind with negligible affinity to classical opioid receptors, induced upregulation of proinflammatory cytokine and chemokine production in rat isolated dorsal spinal cord. Chronic intrathecal (+)-methadone produced hyperalgesia and allodynia, which were associated with significantly increased spinal glial activation (TLR4 mRNA and protein) and the expression of multiple chemokines and cytokines. Statistical analysis suggests that a cluster of cytokines and chemokines may contribute to these nociceptive behavioral changes. Acute intrathecal (+)-methadone and (+)-morphine were also found to induce microglial, interleukin-1 and TLR4/myeloid differentiation factor-2 (MD-2) dependent enhancement of pain responsivity. In silico docking analysis demonstrated (+)-naloxone sensitive docking of (+)-methadone and (+)-morphine to human MD-2. Collectively, these data provide the first evidence of the pro-nociceptive consequences of small molecule xenobiotic activation of spinal TLR4 signaling independent of classical opioid receptor involvement. [Copyright &y& Elsevier]

Abstract: Morphine-3-glucoronide (M3G) is a major morphine metabolite detected in cerebrospinal fluid of humans receiving systemic morphine. M3G has little-to-no affinity for opioid receptors and induces pain by unknown mechanisms. The pain-enhancing effects of M3G have been proposed to significantly and progressively oppose morphine analgesia as metabolism ensues. We have recently documented that morphine activates toll-like receptor 4 (TLR4), beyond its classical actions on μ-opioid receptors. This suggests that M3G may similarly activate TLR4. This activation could provide a novel mechanism for M3G-mediated pain enhancement, as (a) TLR4 is predominantly expressed by microglia in spinal cord and (b) TLR4 activation releases pain-enhancing substances, including interleukin-1 (IL-1). We present in vitro evidence that M3G activates TLR4, an effect blocked by TLR4 inhibitors, and that M3G activates microglia to produce IL-1. In vivo, intrathecal M3G (0.75 μg) induced potent allodynia and hyperalgesia, blocked or reversed by interleukin-1 receptor antagonist, minocycline (microglial inhibitor), and (+)-and (−)-naloxone. This latter study extends our prior demonstrations that TLR4 signaling is inhibited by naloxone nonstereoselectively. These results with (+)-and (−)-naloxone also demonstrate that the effects cannot be accounted for by actions at classical, stereoselective opioid receptors. Hyperalgesia (allodynia was not tested) and in vitro M3G-induced TLR4 signaling were both blocked by 17-DMAG, an inhibitor of heat shock protein 90 (HSP90) that can contribute to TLR4 signaling. Providing further evidence of proinflammatory activation, M3G upregulated TLR4 and CD11b (microglial/macrophage activation marker) mRNAs in dorsal spinal cord as well as IL-1 protein in the lumbosacral cerebrospinal fluid. Finally, in silico and in vivo data support that the glucuronic acid moiety is capable of inducing TLR4/MD-2 activation and enhanced pain. These data provide the first evidence for a TLR4 and IL-1 mediated component to M3G-induced effects, likely of at least microglial origin. [Copyright &y& Elsevier]

Abstract: Spinal cord microglial toll-like receptor 4 (TLR4) has been implicated in enhancing neuropathic pain and opposing morphine analgesia. The present study was initiated to explore TLR4-mediated pain modulation by intrathecal lipopolysaccharide, a classic TLR4 agonist. However, our initial study revealed that intrathecal lipopolysaccharide failed to induce low-threshold mechanical allodynia in naive rats, suggestive that TLR4 agonism may be insufficient to enhance pain. These studies explore the possibility that a second signal is required; namely, heat shock protein-90 (HSP90). This candidate was chosen for study given its known importance as a regulator of TLR4 signaling. A combination of in vitro TLR4 cell signaling and in vivo behavioral studies of pain modulation suggest that TLR4-enhancement of neuropathic pain and TLR4-suppression of morphine analgesia each likely require HSP90 as a cofactor for the effects observed. In vitro studies revealed that dimethyl sulfoxide (DMSO) enhances HSP90 release, suggestive that this may be a means by which DMSO enhances TLR4 signaling. While 2 and 100 μg lipopolysaccharide intrathecally did not induce mechanical allodynia across the time course tested, co-administration of 1 μg lipopolysaccharide with a drug that enhances HSP90-mediated TLR4 signaling now induced robust allodynia. In support of this allodynia being mediated via a TLR4/HSP90 pathway, it was prevented or reversed by intrathecal co-administration of a HSP90 inhibitor, a TLR4 inhibitor, a microglia/monocyte activation inhibitor (as monocyte-derived cells are the predominant cell type expressing TLR4), and interleukin-1 receptor antagonist (as this proinflammatory cytokine is a downstream consequence of TLR4 activation). Together, these results suggest for the first time that TLR4 activation is necessary but not sufficient to induce spinally mediated pain enhancement. Rather, the data suggest that TLR4-dependent pain phenomena may require contributions by multiple components of the TLR4 receptor complex. [Copyright &y& Elsevier]

Tennis is a popular racquet sport played by boys, girls, men and women. Tennis players frequently begin playing in childhood and may continue playing into late adulthood. Preadolescent and adolescent players have open growth plates and a reduced muscle power, lower level of coordination and smaller stature compared with adult players. The physical characteristics of the young tennis player mean that unique demands are placed on the developing athlete which can, in turn, be associated with different types and patterns of injury. The most common types of injury in tennis players of all ages are muscle sprains and ligament sprains secondary to overuse. These are a particular problem in the adolescent age group because, in general, this group begin playing with a lower level of physical conditioning. Fortunately, injuries in younger players are usually not longstanding and the overuse (chronic) problems seen in older players, such as patellar tendinosis and tennis elbow, are less common in younger players. Anatomically, lower extremity injuries are twice as common as those to the upper extremity or spine, with ankle injury being the most common. Prevention of injury in young tennis players, or at least a reduction in the incidence, is possible. Some traumatic injuries, including contusions, abrasions, lacerations and fractures, may be unavoidable as a result of aggressive play, but others may be prevented by monitoring equipment and the court surface to ensure a safe field of play. The prime target of prevention in young tennis players should be overuse injuries. The principles of ‘overload’ and staged involvement are of particular importance in this age group. A gradual, progressive increase in the intensity of tennis practice, the slow introduction of new court surfaces and a staged progression in the teaching of tennis skills can help to reduce the incidence of injury in young tennis players. [ABSTRACT FROM AUTHOR]

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at toll-like receptor 4 (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 nuclear factor kappaB (NF-κB), increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E 2 (PGE 2 ) from these cells. (−)-Morphine-induced upregulation of TNFα mRNA and PGE 2 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 sequelae. 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. [ABSTRACT FROM AUTHOR]