Wen-Wu Li

Post-junctional facilitation of Substance P signaling in a tibia fracture rat model of complex regional pain syndrome type I.

ABSTRACT Tibia fracture in rats evokes nociceptive, vascular, and bone changes resembling complex regional pain syndrome (CRPS). Substance P (SP) signaling contributes to the hindpaw warmth, increased vascular permeability, and edema observed in this model, suggesting that neurogenic inflammatory responses could be enhanced after fracture. Four weeks after tibia fracture we measured SP and calcitonin gene‐related peptide (CGRP) protein levels in the sciatic nerve and serum. Hindpaw skin extravasation responses and SP receptor (NK1), CGRP receptor (calcitonin receptor‐like receptor, CRLR) and neutral endopeptidase (NEP) protein levels were also determined. Gene expression levels of these peptides, receptors, and peptidase were examined in the DRG and skin. Spontaneous and intravenous SP‐evoked extravasation responses were increased ipsilateral, but not contralateral to the fracture. Fracture increased SP and CGRP gene expression in the ipsilateral L4,L5 DRG and neuropeptide protein levels in the sciatic nerve and in serum, but had no effect on electrically evoked SP and CGRP release. NK1 receptor expression was increased in the ipsilateral hindpaw skin keratinocytes and endothelial cells after injury, but CRLR and NEP expression were unchanged. Fracture also increased epidermal thickness, but had no effect on epidermal skin neurite counts. These results demonstrate that spontaneous and intravenous SP‐evoked extravasation responses are enhanced in the ipsilateral hindlimb after fracture and that fracture chronically increases the expression of endothelial and keratinocyte NK1 receptors in the injured limb. We postulate that SP activation of these up‐regulated NK1 receptors results in skin warmth, protein leakage, edema, and keratinocyte proliferation in the injured limb.

The role of enhanced cutaneous IL-1β signaling in a rat tibia fracture model of complex regional pain syndrome

ABSTRACT Tibia fracture in rats initiates a syndrome resembling the complex regional pain syndrome type I. Accumulating evidence indicates that IL‐1β is involved in the modulation of nociceptive information and it acts as an intermediate inflammatory mediator via up‐regulation of NGF. We hypothesized that IL‐1β signaling might mediate the development of the CRPS‐like changes after tibial fracture, either directly or by stimulating NGF expression. Rats underwent distal tibia fracture and casting for 4 weeks and were chronically treated with an IL‐1 receptor antagonist (IL‐1ra). Nociceptive testing and assessment of edema and hindpaw warmth were performed at baseline and after cast removal. Bone microarchitecture was evaluated by micro‐computed tomography. Confocal immunofluorescence and in situ hybridization techniques were used to evaluate changes in the cutaneous expression of IL‐1β at 4 weeks post‐fracture. The nociceptive and vascular effects of intraplantar IL‐1β injections were evaluated in intact rats at different time points after injection. We found that: (1) IL‐1ra reduced fracture‐induced nociceptive sensitization, but did not decrease hindpaw edema or warmth, (2) fracture chronically up‐regulated IL‐1β mRNA and protein expression in hindpaw skin keratinocytes, (3) IL‐1β intraplantar injection induced mechanical allodynia in a dose‐dependent manner and stimulated keratinocyte NGF expression in the hindpaw skin, and (4) intraplantar injection of NGF‐induced nociceptive sensitization. Collectively, these results indicate that cutaneous IL‐1β signaling can contribute to chronic regional nociceptive sensitization after fracture, possibly by stimulating NGF over‐expression in keratinocytes. Our data also highlight the importance of the keratinocyte as the primary source of post‐traumatic IL‐1β over‐expression.

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.

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.

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.

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.

Activation of Cutaneous Immune Responses in Complex Regional Pain Syndrome

UNLABELLED The pathogenesis of complex regional pain syndrome (CRPS) is unresolved, but tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are elevated in experimental skin blister fluid from CRPS-affected limbs, as is tryptase, a marker for mast cells. In the rat fracture model of CRPS, exaggerated sensory and sympathetic neural signaling stimulate keratinocyte and mast cell proliferation, causing the local production of high levels of inflammatory cytokines leading to pain behavior. The current investigation used CRPS patient skin biopsies to determine whether keratinocyte and mast cell proliferation occur in CRPS skin and to identify the cellular source of the up-regulated TNF-α, IL-6, and tryptase observed in CRPS experimental skin blister fluid. Skin biopsies were collected from the affected skin and the contralateral mirror site in 55 CRPS patients and the biopsy sections were immunostained for keratinocyte, cell proliferation, mast cell markers, TNF-α, and IL-6. In early CRPS, keratinocytes were activated in the affected skin, resulting in proliferation, epidermal thickening, and up-regulated TNF-α and IL-6 expression. In chronic CRPS, there was reduced keratinocyte proliferation, leading to epidermal thinning in the affected skin. Acute CRPS patients also had increased mast cell accumulation in the affected skin, but there was no increase in mast cell numbers in chronic CRPS. PERSPECTIVE The results of this study support the hypotheses that CRPS involves activation of the innate immune system, with keratinocyte and mast cell activation and proliferation, inflammatory mediator release, and pain.

Epigenetic Regulation of Spinal CXCR2 Signaling in Incisional Hypersensitivity in Mice

Background: The regulation of gene expression in nociceptive pathways contributes to the induction and maintenance of pain sensitization. Histone acetylation is a key epigenetic mechanism controlling chromatin structure and gene expression. Chemokine CC motif receptor 2 (CXCR2) is a proinflammatory receptor implicated in neuropathic and inflammatory pain and is known to be regulated by histone acetylation in some settings. The authors sought to investigate the role of histone acetylation on spinal CXCR2 signaling after incision. Methods: Groups of 5–8 mice underwent hind paw incision. Suberoylanilide hydroxamic acid and anacardic acid were used to inhibit histone deacetylase and histone acetyltransferase, respectively. Behavioral measures of thermal and mechanical sensitization as well as hyperalgesic priming were used. Both message RNA quantification and chromatin immunoprecipitation analysis were used to study the regulation of CXCR2 and ligand expression. Finally, the selective CXCR2 antagonist SB225002 was administered intrathecally to reveal the function of spinal CXCR2 receptors after hind paw incision. Results: Suberoylanilide hydroxamic acid significantly exacerbated mechanical sensitization after incision. Conversely, anacardic acid reduced incisional sensitization and also attenuated incision-induced hyperalgesic priming. Overall, acetylated histone H3 at lysine 9 was increased in spinal cord tissues after incision, and enhanced association of acetylated histone H3 at lysine 9 with the promoter regions of CXCR2 and keratinocyte-derived chemokine (CXCL1) was observed as well. Blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision. Conclusions: Histone modification is an important epigenetic mechanism regulating incision-induced nociceptive sensitization. The spinal CXCR2 signaling pathway is one epigenetically regulated pathway controlling early and latent sensitization after incision.

Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome

BackgroundDistal limb fracture in man can induce a complex regional pain syndrome (CRPS) with pain, warmth, edema, and cutaneous inflammation. In the present study substance P (SP, Tac1−/−) and CGRP receptor (RAMP1−/−) deficient mice were used to investigate the contribution of neuropeptide signaling to CRPS-like changes in a tibia fracture mouse model. Wildtype, Tac1−/−, and RAMP1−/− mice underwent tibia fracture and casting for 3 weeks, then the cast was removed and hindpaw mechanical allodynia, unweighting, warmth, and edema were tested over time. Hindpaw skin was collected at 3 weeks post-fracture for immunoassay and femurs were collected for micro-CT analysis.ResultsWildtype mice developed hindpaw allodynia, unweighting, warmth, and edema at 3 weeks post-fracture, but in the Tac1−/− fracture mice allodynia and unweighting were attenuated and there was no warmth and edema. RAMP1−/− fracture mice had a similar presentation, except there was no reduction in hindpaw edema. Hindpaw skin TNFα, IL-1β, IL-6 and NGF levels were up-regulated in wildtype fracture mice at 3 weeks post-fracture, but in the Tac1−/− and RAMP1−/− fracture mice only IL-6 was increased. The epidermal keratinocytes were the cellular source for these inflammatory mediators. An IL-6 receptor antagonist partially reversed post-fracture pain behaviors in wildtype mice.ConclusionsIn conclusion, both SP and CGRP are critical neuropeptide mediators for the pain behaviors, vascular abnormalities, and up-regulated innate immune responses observed in the fracture hindlimb. We postulate that the residual pain behaviors observed in the Tac1−/− and RAMP1−/− fracture mice are attributable to the increased IL-6 levels observed in the hindpaw skin after fracture.

Keratinocyte expression of inflammatory mediators plays a crucial role in substance P-induced acute and chronic pain

Tibia fracture in rats followed by cast immobilization leads to nociceptive, trophic, vascular and bone-related changes similar to those seen in Complex Regional Pain Syndrome (CRPS). Substance P (SP) mediated neurogenic inflammation may be responsible for some of the signs of CRPS in humans. We therefore hypothesized that SP acting through the SP receptor (NK1) leads to the CRPS-like changes found in the rat model. In the present study, we intradermally injected rats with SP and monitored hindpaw mechanical allodynia, temperature, and thickness as well as tissue levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and nerve growth factor-β (NGF) for 72 h. Anti-NGF antibody was utilized to block the effects of SP-induced NGF up-regulation. Fracture rats treated with the selective NK1 receptor antagonist LY303870 prior to cast removal were assessed for BrdU, a DNA synthesis marker, incorporation in skin cells to examine cellular proliferation. Bone microarchitecture was measured using micro computed tomography (μCT). We observed that: (1) SP intraplantar injection induced mechanical allodynia, warmth and edema as well as the expression of nociceptive mediators in the hindpaw skin of normal rats, (2) LY303870 administered intraperitoneally after fracture attenuated allodynia, hindpaw unweighting, warmth, and edema, as well as cytokine and NGF expression, (3) LY303870 blocked fracture-induced epidermal thickening and BrdU incorporation after fracture, (4) anti-NGF antibody blocked SP-induced allodynia but not warmth or edema, and (5) LY303870 had no effect on bone microarchitecture. Collectively our data indicate that SP acting through NK1 receptors supports the nociceptive and vascular components of CRPS, but not the bone-related changes.