Xiangqi Li

A murine model of opioid-induced hyperalgesia

Controversies surround the possible long-term physiological and psychological consequences of opioid use. Analgesic tolerance and addiction are commonly at the center of these controversies, but other concerns exist as well. A growing body of evidence suggests that hyperalgesia caused by the chronic administration of opioids can occur in laboratory animals and in humans. In these studies we describe a murine model of opioid-induced hyperalgesia (OIH). After the treatment of mice for 6 days with implanted morphine pellets followed by their removal, both thermal hyperalgesia and mechanical allodynia were documented. Additional experiments demonstrated that prior morphine treatment also increased formalin-induced licking behavior. These effects were intensified by intermittent abstinence accomplished through administration of naloxone during morphine treatment. Experiments designed to determine if the mu-opioid receptor mediated OLH in our model revealed that the relatively-selective mu-opioid receptor agonist fentanyl induced the thermal hyperalgesia and mechanical allodynia characteristic of OIH when administered in intermittent boluses over 6 days. In complimentary experiments we found that CXBK mice which have reduced mu-opioid receptor binding displayed no significant OIH after morphine treatment. Finally, we explored the pharmacological sensitivities of OIH. We found that the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the heme oxygenase (HO) inhibitor tin protoporphyrin (Sn-P) dose-dependently reduced OIH in this model while the NSAID indomethacin had no effect. Thus we have characterized a murine model of OIH which will be useful in the pursuit of the molecular mechanisms underlying this phenomenon.

Opioid-Induced Hyperalgesia and Incisional Pain

Opioids occupy a position of unsurpassed clinical utility in the treatment of pain of many etiologies. However, recent reports in laboratory animals and humans have documented the occurrence of hyperalgesia when the administration of opioids is abruptly tapered or discontinued, a condition known as opioid-induced hyperalgesia (OIH). In these studies we documented that rats administered morphine (40 mg · kg−1 · day−1 for 6 days) via subcutaneous osmotic minipumps demonstrated thermal hyperalgesia and mechanical allodynia for several days after the cessation of morphine administration. Additional experiments using a rat model of incisional pain showed that that attributable to OIH were additive with the hyperalgesia and allodynia that resulted from incision. In our final experiments we observed that if naloxone is administered chronically before incision then discontinued (20 mg · kg−1 · day−1 for 6 days), the hyperalgesia and allodynia that result from hind paw incision was markedly reduced. In contrast, naloxone 1 mg/kg administered acutely after hind paw incision increased hyperalgesia and allodynia. We conclude that the chronic administration of exogenous opioid receptor agonists and antagonists before incision can alter the hyperalgesia and allodynia observed in this pain model, perhaps by altering intrinsic opioidergic systems involved in setting thermal and mechanical nociceptive thresholds.

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.

Effect of anti-NGF antibodies in a rat tibia fracture model of complex regional pain syndrome type I

&NA; Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia resembling the clinical characteristics of patients with complex regional pain syndrome type I (CRPS I). Nerve growth factor (NGF) has been shown to support nociceptive and other types of changes found in neuropathic pain models. We hypothesized that anti‐NGF antibodies might reduce one or more of the CRPS I‐like features of the rat fracture model. For our studies one distal tibia of each experimental rat was fractured and casted for 4 weeks. The rats were injected with anti‐NGF or vehicle at days 17 and 24 post‐fracture. Nociceptive testing as well as assessment of edema and hindpaw warmth were followed during this period. Molecular and biochemical techniques were used to follow cytokine, NGF and neuropeptide levels in hindpaw skin and sciatic nerves. Lumbar spinal cord Fos immunostaining was performed. Bone microarchitecture was measured using microcomputed tomography (μCT). We found that tibia fracture upregulated NGF expression in hindpaw skin and tibia bone along with sciatic nerve neuropeptide content. We also found nociceptive sensitization, enhanced spinal cord Fos expression, osteopenia and enhanced cytokine content of hindpaw skin on the side of the fracture. Anti‐NGF treatment reduced neuropeptide levels in sciatic nerve and reduced nociceptive sensitization. There was less spinal cord Fos expression and bone loss in the anti‐NGF treated animals. Conversely, anti‐NGF did not decrease hindpaw edema, warmth or cytokine production. Collectively, anti‐NGF reduced some but not all signs characteristic of CRPS illustrating the complexity of CRPS pathogenesis and NGF signaling.

TNF signaling contributes to the development of nociceptive sensitization in a tibia fracture model of complex regional pain syndrome type I

&NA; Tibia fracture in rats initiates a cascade of nociceptive, vascular, and bone changes resembling complex regional pain syndrome type I (CRPS I). Previous studies suggest that the pathogenesis of these changes is attributable to an exaggerated regional inflammatory response to injury. We postulated that the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF) might mediate the development of CRPS‐like changes after fracture. RT‐PCR and EIA assays were used to evaluate changes in TNF expression and content in skin, nerve, and bone after fracture. Bilateral hindpaw thickness, temperature, and nociceptive thresholds were determined, and bone microarchitecture was measured using microcomputed tomography. Lumbar spinal cord Fos immunostaining was performed for quantification of Fos positive neurons. After baseline testing, the distal tibia was fractured and the hindlimb casted for 4 weeks. The rats were subcutaneously injected either with a soluble TNF receptor type 1 (sTNF‐R1, 5 mg/kg/d) or saline every 3 days over 28 days and then were retested at 4 weeks post‐fracture. Tibia fracture chronically upregulated TNF expression and protein levels in the hindpaw skin and sciatic nerve. After fracture the rats developed hindpaw mechanical allodynia and unweighting, which were reversed by sTNF‐R1 treatment. Consistent with the behavioral data, spinal Fos increased after fracture and this effect was inhibited by sTNF‐R1 treatment. Collectively, these data suggest that facilitated TNF signaling in the hindlimb is an important mediator of chronic regional nociceptive sensitization after fracture, but does not contribute to the hindlimb warmth, edema, and bone loss observed in this CRPS I model.

Fracture induces keratinocyte activation, proliferation, and expression of pro-nociceptive inflammatory mediators

&NA; Tibia fracture in rats results in chronic vascular and nociceptive changes in the injured limb resembling complex regional pain syndrome (CRPS) and up‐regulates expression of interleukin 1&bgr; (IL‐1&bgr;), interleukin IL‐6 (IL‐6), tumor necrosis factor‐&agr; (TNF‐&agr;), and nerve growth factor‐&bgr; (NGF‐&bgr;) in the hindpaw skin. When fractured rats are treated with cytokine or NGF inhibitors nociceptive sensitization is blocked. Because there is no leukocyte infiltration in the hindpaw skin we postulated that resident skin cells produce the inflammatory mediators causing nociceptive sensitization after fracture. To test this hypothesis rats underwent distal tibia fracture and hindlimb casting for 4 weeks, then the hindpaw skin was harvested and immunostained for keratin, cytokines and NGF. BrdU staining was used to evaluate cell proliferation. Hindpaw nociceptive thresholds, edema, and temperature were tested before and up to 96 h after intraplantar injections of IL‐6 and TNF‐&agr;. Tibia fracture caused keratinocyte activation, proliferation, and up‐regulated IL‐1&bgr;, IL‐6, TNF‐&agr; and NGF‐&bgr; protein expression in the hindpaw keratinocytes. Local injections of IL‐6 and TNF‐&agr; induced hindpaw mechanical allodynia lasting for several days and modest increases in temperature and edema. These data indicate that activated keratinocytes proliferate and express IL‐1&bgr;, IL‐6, TNF‐&agr;, and NGF‐&bgr; after fracture and that excess amounts of inflammatory mediators in the skin cause sustained nociceptive sensitization. This is the first study demonstrating in vivo keratinocyte expression of IL‐6, TNF‐&agr; and NGF‐&bgr; in a CRPS model and we postulate that the keratinocyte is the primary cellular source for the inflammatory signals mediating cutaneous nociceptive sensitization in early CRPS.

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.

Blockade of the Complement C5a Receptor Reduces Incisional Allodynia, Edema, and Cytokine Expression

Background:Activation of the complement system is one component of the inflammatory response. Various components of the complement system participate in killing foreign organisms, recruiting immune cells, enhancing edema, and stimulating cytokine formation. Complement-mediated enhancement of the inflammation surrounding surgical incisions may increase pain. Methods:In these studies, the authors used a murine hind paw incisional model to study the role of the complement C5a receptor in supporting incisional inflammation. At baseline and at various time points after incision, they measured the effects of a highly selective C5a receptor antagonist on nociceptive thresholds, edema formation, and cytokine production in the skin surrounding the incision. They also measured changes in C5a receptor expression near the incisions. Results:The once-daily injection of the C5a receptor antagonist AcF-[OPdChaWR] reduced mechanical allodynia and edema in the incised hind paw. A multiplexed cytokine assay revealed that 8 of the 18 cytokines examined showed significant increases in skin tissue abundance after incision. Distinct time courses for the patterns of elevation were seen, though some degree of resolution occurred for all cytokines within 96 h. For 7 of these 8 cytokines, the C5a receptor antagonist reduced the enhancement of expression. In addition, the authors found that the C5a receptor messenger RNA level increased 15-fold in the skin surrounding the incisions within 24 h and then slowly declined. Conclusions:The tissue directly surrounding incisions in mouse hind paws undergoes large changes in the content of specific cytokines in addition to demonstrating edema and nociceptive sensitization. By blocking the receptor for one component of the complement system, C5a, all of these changes can be reduced. Complement receptor inhibitors may constitute a novel group of compounds useful in reducing the pain and swelling of surgical incisions.