Abstract
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.
Highlights
Complex regional pain syndrome (CRPS) is a painful, disabling and often chronic condition affecting the extremities and is a frequent sequela of tibial and radial fractures [1]
tumor necrosis factor (TNF)-α expression was upregulated at 1 h post-injection, interleukin 6 (IL-6) and interleukin 1β (IL-1β) levels were increased at 1 h and 3 h post-injection, respectively, and nerve growth factor (NGF) expression gradually increased from 1 h to 24 h post-injection, resolving by 48 h
When the hindpaw skin was harvested at various time points after Substance P (SP) (25 μg) injection for immunostaining with IL-1β antibody, increased IL-1β protein was observed in keratinocytes at 1 h and 3 h after SP injection, resolving by 6 h, which corresponds to the time course observed using EIA assays on skin homogenates (Figure 3)
Summary
Complex regional pain syndrome (CRPS) is a painful, disabling and often chronic condition affecting the extremities and is a frequent sequela of tibial and radial fractures [1]. We described a distal tibial fracture model in rats that exhibits chronic unilateral hindlimb warmth, edema, facilitated spontaneous protein extravasation, allodynia, postural unweighting, and periarticular osteoporosis [2]. These post-fracture changes closely resemble the clinical presentation of patients with acute CRPS. Treating fractured rats with a TNF-α inhibitor (etanercept), an IL-1 receptor antagonist (anakinra), or an anti-NGF antibody (tanezumab) reduced hindpaw allodynia and unweighting at 4 weeks post-fracture [4,5,7] These data indicate that fractureinduced allodynia can be attributed partially to local inflammatory mediators because all these drugs are large molecular weight proteins that cannot cross the blood brain barrier. We identified keratinocytes in the fracture-affected dorsal hindpaw as the primary cellular source of the inflammatory nociceptive mediators TNF-α, IL-1β, IL-6, and NGF in the rat fracture CRPS model [8]
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