De-Yong Liang

Morphine reduces local cytokine expression and neutrophil infiltration after incision

BackgroundInflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines.ResultsA murine incisional model was used to measure the effects of acute morphine administration (0.1–10 mg/kg) on nociceptive thresholds, neutrophil infiltration and cytokine production in hind paw skin 30 minutes and 2 hours after incision. Incised hind paws displayed profound allodynia which was reduced by morphine (0.1–10 mg/kg) in the 2 hours following incision. Skin samples harvested from these mice showed enhanced levels of 5 cytokines: IL-1β, IL-6, tumor necrosis factor alpha (TNFα), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC). Morphine reduced these incision-stimulated levels. Separate analyses measuring myeloperoxidase (MPO) and using immunohistochemistry demonstrated that morphine dose-dependently reduced the infiltration of neutrophils into the peri-incisional tissue. The dose of morphine required for reduction of cytokine accumulation, however, was below that required for inhibition of peri-incisional neutrophil infiltration. Additional immunohistochemical studies revealed wound edge keratinocytes as being an important source of cytokines in the acute phase after incision.ConclusionAcute morphine administration of doses as low as 0.1 mg/kg reduces peri-incisional cytokine expression. A reduction in neutrophil infiltration does not provide a complete explanation for this effect, and keratinocytes may be responsible for some incision area cytokine production. These studies suggest that morphine may alter the inflammatory milieu of incisional wounds, but these alterations do not likely contribute significantly to analgesia in the acute setting.

A genetic analysis of opioid-induced hyperalgesia in mice.

Background:Opioid-induced hyperalgesia (OIH) is a syndrome of increased sensitivity to noxious stimuli, seen after both the acute and chronic administration of opioids, that has been observed in humans and rodent models. This syndrome may reduce the clinical utility of opioids in treating acute and chronic pain. Methods:In these studies, the authors measured the propensity of 15 strains of inbred mice to develop mechanical manifestations of OIH. These data were subjected to in silico genetic analysis, which resulted in the association of haplotypic blocks within or near several known genes. Both pharmacologic agents and transgenic mice were used to confirm the functional association of the most strongly linked gene with OIH. Results:Both baseline mechanical nociceptive thresholds and the percentage changes in these thresholds after 4 days of morphine treatment were found to be highly strain dependent. The haplotypic blocks most strongly associated with the mechanical OIH data were located within the β2 adrenergic receptor gene (β2-AR). Using the selective β2-AR antagonist butoxamine, the authors observed a dose-dependent reversal of OIH. Furthermore, deletion of the β2-AR gene sharply reduced the mechanical allodynia present after morphine treatment in the wild-type mouse strain. Analysis of the associated β2-AR haplotypic block identified single nucleotide polymorphisms potentially explaining in part the strain specific differences in OIH. Conclusions:Genetic variants of the β2-AR gene seem to explain some part of the differences between various strains of mice to develop OIH. The association of this gene with OIH suggests specific pharmacologic strategies for reducing the impact of OIH on patients consuming opioids.

Chronic morphine administration enhances nociceptive sensitivity and local cytokine production after incision.

Background -The chronic use of opioids prior to surgery leads to lowered pain thresholds and exaggerated pain levels after these procedures. Several mechanisms have been proposed to explain this heightened sensitivity commonly termed opioid-induced hyperalgesia (OIH). Most of these proposed mechanisms involve plastic events in the central or peripheral nervous systems. Alterations in the abundance of peripheral mediators of nociception have not previously been explored.Results -In these experiments mice were treated with saline (control) or ascending daily doses of morphine to generate a state of OIH followed by hind paw incision. In other experiments morphine treatment was initiated at the time of incision. Both mechanical allodynia and peri-incisional skin cytokine levels were measured. Myeloperoxidase (MPO) assays were used to determine neutrophil activity near the wounds. The cytokine production inhibitor pentoxifylline was used to determine the functional significance of the excess cytokines in previously morphine treated animals. Mice treated chronically treated with morphine prior to incision were found to have enhanced skin levels of IL-1β, IL-6, G-CSF, KC and TNFα after incision at one or more time points compared to saline pretreated controls. The time courses of individual cytokines followed different patterns. There was no discernable effect of chronic morphine treatment on wound area neutrophil infiltration. Pentoxifylline reduced cytokine levels and reversed the excess mechanical sensitization caused by chronic morphine administration prior to incision. Morphine treatment initiated at the time of incision did not lead to a generalized enhancement of cytokine production or nociceptive sensitization in excess of the levels observed after incision alone.Conclusion -The enhanced level of nociceptive sensitization seen after incision in animals chronically exposed to morphine is associated with elevated levels of several cytokines previously reported to be relevant to this incisional pain model. The cytokines may be functional in supporting nociceptive sensitization because pentoxifylline reverses both peri-incisional skin cytokine levels and OIH. Opioid administration beginning at the time of incision does not seem to have the same cytokine enhancing effect. Approaches to postoperative pain control involving a reduction of cytokines may be an effective way to control excessive pain in patients chronically using opioids prior to surgical procedures.

Substance P Signaling Controls Mast Cell Activation, Degranulation, and Nociceptive Sensitization in a Rat Fracture Model of Complex Regional Pain Syndrome

Background: Patients with complex regional pain syndrome have increased tryptase in the skin of the affected extremity indicating mast cell (MC) accumulation and degranulation, processes known to be mediated by substance P (SP). The dysregulation of SP release from primary afferent neurons is characteristic of complex regional pain syndrome. The authors hypothesized that SP acting through the neurokinin-1 receptor results in mast cell accumulation, degranulation, and nociceptive sensitization in a rat model of complex regional pain syndrome. Methods: Groups of 6–10 rats underwent tibia fracture and hind limb casting for 4 weeks, and the hind paw skin was harvested for histologic and immunohistochemical analysis. The effects of a selective neurokinin-1 receptor antagonist (LY303870) and of direct SP intraplantar injection were measured. Dermal MC degranulation induced by sciatic nerve stimulation and the effects of LY303870 on this process were investigated. Finally, the antinociceptive effects of acute and chronic treatment with a MC degranulator (48/80) were tested. Results: The authors observed that fracture caused MC accumulation, activation, and degranulation, which were inhibited by LY303870; the percentage of MCs in close proximity to peptidergic nerve fibers increased after fracture; electrical stimulation caused MC activation and degranulation, which was blocked by LY303870; intraplantar SP-induced MC degranulation and acute administration of 48/80 caused MC degranulation and enhanced postfracture nociception, but MC-depleted animals showed less sensitization. Conclusions: These results indicate that facilitated peptidergic neuron-MC signaling after fracture can cause MC accumulation, activation, and degranulation in the injured limb, resulting in nociceptive sensitization.

Epigenetic Regulation of Opioid-Induced Hyperalgesia, Dependence and Tolerance in Mice

UNLABELLED Repeated administration of opioids such as morphine induces persistent behavioral changes including opioid-induced hyperalgesia (OIH), tolerance, and physical dependence. In the current work we explored how the balance of histone acetyltransferase (HAT) versus histone deacetylase (HDAC) might regulate these morphine-induced changes. Nociceptive thresholds, analgesia, and physical dependence were assessed during and for a period of several weeks after morphine exposure. To probe the roles of histone acetylation, the HAT inhibitor curcumin or a selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was administered daily to groups of animals. Histone acetylation in spinal cord was assessed by Western blot and immunohistochemistry. Concurrent administration of curcumin with morphine for 4 days significantly reduced development of opioid-induced mechanical allodynia, thermal hyperalgesia, tolerance, and physical dependence. Conversely, the HDAC inhibitor SAHA enhanced these responses. Interestingly, SAHA treatment after the termination of opioid administration sustained these behavioral changes for at least 4 weeks. Histone H3 acetylation in the dorsal horn of the spinal cord was increased after chronic morphine treatment, but H4 acetylation was unchanged. Moreover, we observed a decrease in HDAC activity in the spinal cords of morphine-treated mice while overall HAT activity was unchanged, suggesting a shift toward a state of enhanced histone acetylation. PERSPECTIVE The current study indicates that epigenetic mechanisms play a crucial role in opioid-induced long-lasting neuroplasticity. These results provide new sight into understanding the mechanisms of opioid-induced neuroplasticity and suggest new strategies to limit opioid abuse potential and increase the value of these drugs as analgesics.

Increased expression of Ca2+/calmodulin-dependent protein kinase IIα during chronic morphine exposure

The chronic administration of morphine and related opioid drugs results in tolerance and dependence which limits the clinical utility of these agents. Neuronal plasticity is probably responsible in large part for tolerance and dependence. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in the neuroplastic events underlying memory formation and other phenomena. However, the role of this kinase in morphine tolerance remains unclear. To clarify this issue we explored mRNA and protein expression of CaMKIIalpha in spinal cord tissue from control and morphine treated mice using real-time polymerase chain reaction, Western blot analysis and confocal microscopy. Our chronic exposure paradigm involved the subcutaneous implantation of morphine pellets for 6 days prior to tissue analysis. The results indicate that the levels of CaMKIIalpha mRNA and protein were robustly increased in spinal cord tissue from morphine-treated mice. Confocal microscopy demonstrated that the increase in CaMKIIalpha expression was primarily localized to superficial laminae of the dorsal horn. In addition, the abundance of phosphorylated CaMKIIalpha was increased in spinal cord tissue from morphine-treated mice. We conclude that enhanced CaMKIIalpha expression and activity in spinal cord tissue may contribute to the development of morphine tolerance in mice. The involvement of this enzyme in opioid tolerance suggests other parallels may exist between the neuroplastic events related to memory formation and those related to opioid tolerance or pain.

From mouse to man : the 5-HT3 receptor modulates physical dependence on opioid narcotics

Objectives Addiction to opioid narcotics represents a major public health challenge. Animal models of one component of addiction, physical dependence, show this trait to be highly heritable. The analysis of opioid dependence using contemporary in-silico techniques offers an approach to discover novel treatments for dependence and addiction. Methods In these experiments, opioid withdrawal behavior in 18 inbred strains of mice was assessed. Mice were treated for 4 days with escalating doses of morphine before the administration of naloxone allowing the quantification of opioid dependence. After haplotypic analysis, experiments were designed to evaluate the top gene candidate as a modulator of physical dependence. Behavioral studies as well as measurements of gene expression on the mRNA and protein levels were completed. Finally, a human model of opioid dependence was used to quantify the effects of the 5-HT3 antagonist ondansetron on signs and symptoms of withdrawal. Results The Htr3a gene corresponding to the 5-HT3 receptor emerged as the leading candidate. Pharmacological studies using the selective 5-HT3 antagonist ondansetron supported the link in mice. Morphine strongly regulated the expression of the Htr3a gene in various central nervous system regions including the amygdala, dorsal raphe, and periaqueductal gray nuclei, which have been linked to opioid dependence in previous studies. Using an acute morphine administration model, the role of 5-HT3 in controlling the objective signs of withdrawal in humans was confirmed. Conclusion These studies show the power of in-silico genetic mapping, and reveal a novel target for treating an important component of opioid addiction.

The NALP1 inflammasome controls cytokine production and nociception in a rat fracture model of complex regional pain syndrome.

ABSTRACT Tibia fracture followed by limb immobilization in rats evokes nociceptive and vascular changes resembling complex regional pain syndrome type I (CRPS I). Previously we observed that substance P (SP) and interleukin‐1β (IL‐1β) signaling contribute to chronic regional nociceptive sensitization in this model. It is known that inflammasome multi‐protein complexes containing caspase‐1 and NALP1 are involved in the activation of the IL‐1β family of pro‐nociceptive cytokines expressed in skin and other tissues. Therefore, we hypothesized that SP activated inflammasomes might contribute to mechanical allodynia after fracture. Using this model we observed that: (1) inflammasome components and products NALP1, caspase‐1, IL‐1β and IL‐18 were present in low levels in normal skin, but expression of all these was strongly up‐regulated after fracture, (2) NALP1, caspase‐1 and IL‐1β were co‐expressed in keratinocytes, and the number of NALP1, caspase‐1, and IL‐1β positive cells dramatically increased at 4 weeks post‐fracture, (3) LY303870, an NK1 receptor antagonist, effectively blocked fracture‐induced up‐regulation of activated inflammasome components and cytokines, (4) IL‐1β and IL‐18 intraplantar injection induced mechanical allodynia in normal rats, and (5) both a selective caspase‐1 inhibitor and an IL‐1 receptor antagonist attenuated fracture‐induced hindpaw mechanical allodynia. Collectively, these data suggest that NALP1 containing inflammasomes activated by NK1 receptors are expressed in keratinocytes and contribute to post‐traumatic regional nociceptive sensitization. These findings highlight the possible importance of neuro‐cutaneous signaling and innate immunity mechanisms in the development of CRPS.

Chronic pain and genetic background interact and influence opioid analgesia, tolerance, and physical dependence

Abstract Opioids are commonly used in the treatment of moderate to severe pain. However, their chronic use is limited by analgesic tolerance and physical dependence. Few studies have examined how chronic pain affects the development of tolerance or dependence, and essentially no studies have looked at the role of both genetics and pain together. For these studies we used 12 strains of inbred mice. Groups of mice from each strain were tested at baseline for morphine analgesic sensitivity, mechanical nociceptive threshold, and thermal nociceptive threshold. Mice were then given morphine in a 4‐day escalating morphine administration paradigm followed by reassessment of the morphine dose–response relationship. Finally, physical dependence was measured by administering naloxone. Parallel groups of mice underwent hind paw injection of complete Freund’s adjuvant (CFA) to induce chronic hind paw inflammation 7 days prior to the beginning of testing. The data showed that CFA treatment tended to lower baseline ED50 values for morphine and enhanced the degree of analgesic tolerance observed after 4 days of morphine treatment. In addition, the degree of jumping behavior indicative of physical dependence was often altered if mice had been treated with CFA. The influence of background strain was substantial for all traits measured. In silico haplotypic mapping of the tolerance and physical dependence data demonstrated that CFA pretreatment altered the pattern of the predicted associations and greatly reduced their statistical significance. We conclude that chronic inflammatory pain and genetics interact to modulate the analgesic potency of morphine, tolerance, and physical dependence.

The Role of Interleukin-1 in Wound Biology. Part II: In Vivo and Human Translational Studies

BACKGROUND: In the accompanying paper, we demonstrate that genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production through altering the amount of interleukin (IL)-1 produced. We further investigate the role of IL-1 in incisional wound biology and its effect on wound chemokine production in vivo and whether this mechanism could be active in human subjects. METHODS: A well-characterized murine model of incisional wounding was used to assess the in vivo role of IL-1 in wound biology. The amount of 7 different cytokines/chemokines produced within an experimentally induced skin incision on a mouse paw and the nociceptive response was analyzed in mice treated with an IL-1 inhibitor. We also investigated whether human IL-1&bgr; or IL-1&agr; stimulated the production of chemokines by primary human keratinocytes in vitro, and whether there was a correlation between IL-1&bgr; and chemokine levels in 2 experimental human wound paradigms. RESULTS: Administration of an IL-1 receptor antagonist to mice decreased the nociceptive response to an incisional wound, and reduced the production of multiple inflammatory mediators, including keratinocyte-derived chemokine (KC) and macrophage inhibitory protein (MIP)-1&agr;, within the wounds. IL-1&agr; and IL-1&bgr; stimulated IL-8 and GRO-&agr; (human homologues of murine keratinocyte-derived chemokine) production by primary human keratinocytes in vitro. IL-1&bgr; levels were highly correlated with IL-8 in human surgical wounds, and at cutaneous sites of human ultraviolet B-induced sunburn injury. CONCLUSIONS: IL-1 plays a major role in regulating inflammatory mediator production in wounds through a novel mechanism; by stimulating the production of multiple cytokines and chemokines, it impacts clinically important aspects of wound biology. These data suggest that administration of an IL-1 receptor antagonist within the perioperative period could decrease postsurgical wound pain.