Xiaoyou Shi

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.

Calcitonin gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-κB activation, osteoclastogenesis and bone resorption

Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin-gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP(+)) multinucleated cells, pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-kappaB (NF-kappaB) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10(-10)-10(-8) M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10(-8) M) inhibited receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP(+) cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-kappaB activation, osteoclastogenesis, and bone resorption.

Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro

INTRODUCTION SP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis. MATERIALS AND METHODS Osteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP(+)), measuring pit erosion area, and evaluating RANKL protein production and NF-kappaB activity with ELISA and western blot. RESULTS The NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10(-12) M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10(-8) M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-kappaB in BMMs and directly facilitate RANKL-induced macrophage osteoclastogenesis and bone resorption activity. CONCLUSIONS NK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in vitro. SP neurotransmitter release from sensory neurons could potentially regulate local bone turnover in vivo.

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.

Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision.

ABSTRACT Substance P (SP) signaling facilitates nociceptive sensitization in various inflammatory and chronic pain models and we postulated that SP signaling might also contribute to the development of post‐incisional hyperalgesia. These studies used mice with a deletion of the pre‐protachykinin A gene (ppt‐A−/−) which codes for SP to determine the role of SP signaling in post‐incisional pain and in the increased cytokine and nerve growth factor (NGF) expression observed in the incised skin. SP deficient ppt‐A−/− mice displayed reduced mechanical allodynia and heat hyperalgesia compared to the wild‐type (wt) mice at all post‐incision time points, despite similar baseline values (p < 0.001). Furthermore, the NK‐1 receptor antagonist LY303870 attenuated mechanical allodynia produced by incision in the wt mice (p < 0.001). Incision also up‐regulated IL‐6, TNF‐&agr; and KC levels but not IL‐1&bgr; after 2 h in the wt mice skin. However, ppt‐A−/− mice had more skin NGF levels 2 h post‐incision. Subcutaneous hind paw SP injection produced acute and transient elevations of IL‐1&bgr;, IL‐6, and KC but modest elevations in TNF‐&agr; levels in the wt mice. Systemic LY303870 reversed the SP‐induced elevations of these cytokines. Hind paw injection of IL‐6 and NGF dose dependently produced less mechanical allodynia in the ppt‐A−/− compared to wt mice. Additionally, SP produced mechanical allodynia in a dose‐dependent fashion in wt mice. Therefore, SP supports nociceptive sensitization after hind paw incision and potentially participates directly in modulating the intensity of inflammatory response in peri‐incisional tissue.

Pentoxifylline attenuates nociceptive sensitization and cytokine expression in a tibia fracture rat model of complex regional pain syndrome

Background: Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia, a syndrome resembling complex regional pain syndrome (CRPS). Previous studies suggest that the pathogenesis of some of these changes involves an exaggerated regional inflammatory response to injury and we postulated that inflammatory cytokines contribute to the development of CRPS‐like changes after fracture.

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.

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.

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.