Alpha-7 Nicotinic Acetylcholine Receptor Activation Inhibits Trauma Induced Pronociceptive Autoimmune Responses.

Both autonomic nervous system dysfunction and immune system activation are characteristic of chronic pain after limb injuries. Cholinergic agonists reduce immune system activation in many settings. We hypothesized, therefore, that alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist administration would reduce nociceptive and immune changes after tibia fracture and cast immobilization in mice. Fracture mice were treated with either vehicle, a low (.2 mg/kg) dose, or a high (1 mg/kg) dose of the selective α7nAChR agonist PNU-282987 for 4 weeks. We assessed hindpaw allodynia and weight bearing as behavioral outcomes. The assessment of adaptive immune responses included regional lymph node hypertrophy, germinal center formation, α7nAChR expression, and IgM deposition. Assessment of innate immune system activation focused on IL-1ß and IL-6 generation in fractured hindlimb skin. We observed that mechanical allodynia and unweighting were alleviated by PNU-282987 treatment. Drug treatment also reduced popliteal lymph node hypertrophy and germinal center formation. Immunohistochemical studies localized α7nAChR to germinal center B lymphocytes, and this expression increased after fracture. Analysis of fracture limb hindpaw skin demonstrated increased inflammatory mediator (IL-1ß and IL-6) levels and IgM deposition, which were abrogated by PNU-282987. Serum analyses demonstrated fracture-induced IgM reactivity against keratin 16, histone 3.2, GFAP, and NMDAR-2B. Administration of PNU-282987 reduced the enhancement of IgM reactivity. Collectively, these data suggest that the α7nAChR is involved in regulating posttraumatic innate and adaptive immune responses and the associated nociceptive sensitization. PERSPECTIVE: These studies evaluate the effects of a selective α7nAChR agonist in a tibial fracture/cast immobilization model of limb pain. Administration of the drug reduced nociceptive and functional changes 4 weeks after injury. These novel findings suggest that well-tolerated α7nAChR agonists may be viable analgesics for chronic pain after limb injuries.

Pronociceptive autoantibodies in the spinal cord mediate nociceptive sensitization, loss of function, and spontaneous pain in the lumbar disk puncture model of chronic back pain.

ABSTRACT: Previously, we observed that B cells and autoantibodies mediated chronic nociceptive sensitization in the mouse tibia fracture model of complex regional pain syndrome and that complex regional pain syndrome patient antibodies were pronociceptive in fracture mice lacking mature B cells and antibodies (muMT). The current study used a lumbar spinal disk puncture (DP) model of low back pain in wild-type (WT) and muMT mice to evaluate pronociceptive adaptive immune responses. Spinal disks and cords were collected 3 weeks after DP for polymerase chain reaction and immunohistochemistry analyses. Wild-type DP mice developed 24 weeks of hindpaw mechanical allodynia and hyperalgesia, grip weakness, and a conditioned place preference response indicative of spontaneous pain, but pain responses were attenuated or absent in muMT DP mice. Spinal cord expression of inflammatory cytokines, immune cell markers, and complement components were increased in WT DP mice and in muMT DP mice. Dorsal horn immunostaining in WT DP mice demonstrated glial activation and increased complement 5a receptor expressionin spinal neurons. Serum collected from WT DP mice and injected into muMT DP mice caused nociceptive sensitization, as did intrathecal injection of IgM collected from WT DP mice, and IgM immune complexes were observed in lumbar spinal disks and cord of WT DP mice. Serum from WT tibia fracture mice was not pronociceptive in muMT DP mice and vice versa, evidence that each type of tissue trauma chronically generates its own unique antibodies and targeted antigens. These data further support the pronociceptive autoimmunity hypothesis for the transition from tissue injury to chronic musculoskeletal pain state.

Dimethyl Fumarate Reduces Oxidative Stress and Pronociceptive Immune Responses in a Murine Model of Complex Regional Pain Syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works through the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional, and immunological changes measured in a model of CRPS. METHODS: Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25 mg/kg/d orally, per os for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments and hindlimb weight-bearing data were collected. The markers of oxidative stress malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1ß and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wildtype fractured mice to B cell-deficient fractured muMT mice (mice lacking B cells and immunoglobulin) helped to assess the pronociceptive activity of humoral factors. RESULTS: We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy, and the accumulation of IgM in fractured limbs. The sera of fractured vehicle-treated but not DMF-treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice. CONCLUSIONS: Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation.

Mice lacking substance P have normal bone modeling but diminished bone formation, increased resorption, and accelerated osteopenia with aging.

Substance P (SP) is a sensory neuropeptide that is expressed by the neurons innervating bone. There is considerable evidence that SP can regulate bone cell function in vitro, but it is unclear whether SP modulates bone modeling or remodeling in vivo. To answer this question we characterized the bone phenotype of mice with deletion of the Tac1 gene expressing SP. The phenotypes of 2-month-old and 5-month-old SP deficient mice and their wildtype controls were characterized by using µCT imaging, static and dynamic bone histomorphometry, and urinary deoxypyridinoline cross-links (DPD) measurement. No differences in bone phenotypes were observed between the 2 strains at 2 months of age. By 5 months both the wildtype and SP deficient mice had developed cancellous osteopenia, but relative to the wild-type mice the SP deficient mice had significantly greater cancellous bone loss. The SP deficient mice also exhibited decreased bone formation, increased osteoclast number, and increased urinary DPD levels. Cortical defect early repair was delayed in 5-month-old mice lacking SP. Collectively, these findings indicate that SP signaling is not required for bone modeling, but SP signaling reduces age-related osteopenia and accelerates cortical defect reparation, data supporting the hypothesis that SP is an anabolic physiologic regulator of bone metabolism.

Complex regional pain syndrome patient immunoglobulin M has pronociceptive effects in the skin and spinal cord of tibia fracture mice.

It has been proposed that complex regional pain syndrome (CRPS) is a post-traumatic autoimmune disease. Previously, we observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture model and that serum immunoglobulin M (IgM) antibodies from fracture mice have pronociceptive effects in muMT fracture mice lacking B cells. The current study evaluated the pronociceptive effects of injecting CRPS patient serum or antibodies into muMT fracture mice by measuring hind paw allodynia and unweighting changes. Complex regional pain syndrome serum binding was measured against autoantigens previously identified in the fracture mouse model. Both CRPS patient serum or IgM antibodies had pronociceptive effects in the fracture limb when injected systemically in muMT fracture mice, but normal subject serum and CRPS patient IgG antibodies had no effect. Furthermore, CRPS serum IgM antibodies had pronociceptive effects when injected into the fracture limb hind paw skin or intrathecally in the muMT fracture mice. Early (1-12 months after injury) CRPS patient (n = 20) sera were always pronociceptive after systemic injection, and chronic (>12 months after injury) CRPS sera were rarely pronociceptive (2/20 patients), while sera from normal subjects (n = 20) and from patients with uncomplicated recoveries from orthopedic surgery and/or fracture (n = 15) were never pronociceptive. Increased CRPS serum IgM binding was observed for keratin 16, histone 3.2, gamma actin, and alpha enolase autoantigens. We postulate that CRPS patient IgM antibodies bind to neoantigens in the fracture mouse skin and spinal cord to initiate a regionally restricted pronociceptive complement response potentially contributing to the CRPS disease process.

Sex differences in the temporal development of pronociceptive immune responses in the tibia fracture mouse model.

Previously, distinct sex differences were observed in the pronociceptive role of spinal immune cells in neuropathic and inflammatory mouse pain models. Both peripheral and central innate and adaptive immune changes contribute to sensitization in the tibia fracture rodent model of complex regional pain syndrome, and the current study evaluated sex differences in the development of pronociceptive immune responses after fracture. At 4 and 7 weeks after fracture, the analgesic effects of a microglia inhibitor were tested in male and female mice, and polymerase chain reaction was used to measure inflammatory mediator expression in skin and spinal cord. The temporal progression of complex regional pain syndrome-like changes in male and female wild-type and muMT fracture mice lacking B cells and antibodies were evaluated, and IgM antibody deposition measured. Pronociceptive effects of injecting wild-type fracture mouse serum into muMT fracture mice were also tested in both sexes, and the role of sex hormones was evaluated in the postfracture development of pronociceptive immune responses. Long-lasting immune changes developed in the fracture limb and corresponding spinal cord of both male and female mice, including upregulated neuropeptide and cytokine signaling, microglial activation, and pronociceptive autoimmunity. These complex postfracture immune responses were sexually dichotomous and interacted in temporally evolving patterns that generated post-traumatic nociceptive sensitization in both sexes lasting for up to 5 months. Unfortunately, the redundancy and plasticity of these chronic post-traumatic immune responses suggest that clinical interventions focusing on any single specific pronociceptive immune change are likely to be ineffectual.

Oxidative Stress Contributes to Fracture/Cast-Induced Inflammation and Pain in a Rat Model of Complex Regional Pain Syndrome.

Clinical evidence suggests that vitamin C (Vit C) may protect against the development of complex regional pain syndrome (CRPS) after fracture or surgery. Tibia fracture followed by 4 weeks of cast immobilization (fracture/cast) in rats results in nociceptive, vascular, and bone changes resembling clinical CRPS. In this study, fracture/cast rats were treated with the oxidative stress inhibitors Vit C, N-acetyl cysteine, or 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl to examine their effects on CRPS-related nociceptive and vascular changes. Administration of these agents significantly reduced fracture/cast-induced cutaneous allodynia by 64 to 78%, muscle hyperalgesia by 34 to 40%, and hind limb unweighting by 48 to 89%. Treatments with Vit C and N-acetyl cysteine reduced the oxidative stress markers malondialdehyde in the skin, muscle, and sciatic nerve, and lactate in the gastrocnemius muscle of the fracture/cast limb. Furthermore, Vit C treatment inhibited the post-fracture upregulation of substance P and calcitonin gene-related peptide in the sciatic nerve and the increased expression of the pain-related inflammatory mediators, including interleukin (IL)-6, and nerve growth factor in the skin and IL-1ß, and IL-6 in the muscle of the post-fracture/cast limb. These data suggest that oxidative stress may contribute to the nociceptive features of the rat CRPS model. PERSPECTIVE: Vit C reduced the CRPS-like signs, oxidative stress, and the upregulation of neuropeptide production and inflammatory mediators observed after tibia fracture and casting in rats. Limiting oxidative stress by use of Vit C or alternative strategies could reduce the risk of developing CRPS after surgery or other forms of trauma.

Passive transfer autoimmunity in a mouse model of complex regional pain syndrome.

It has been proposed that complex regional pain syndrome (CRPS) is a posttraumatic autoimmune disease, and we previously observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture CRPS model. The current study used the mouse model to evaluate the progression of postfracture CRPS-like changes in wild-type (WT) and muMT fracture mice lacking B cells and antibodies. The pronociceptive effects of injecting WT fracture mouse serum antibodies into muMT fracture mice were also evaluated. Postfracture pain behaviors transitioned from being initially dependent on both innate and autoimmune inflammatory mechanisms at 3 weeks after fracture to being entirely mediated by antibody responses at 12 weeks after fracture and spontaneously resolving by 21 weeks after fracture. Furthermore, serum IgM antibodies from WT fracture mice had pronociceptive effects in the fracture limb when injected into muMT fracture mice. IgM antibody levels gradually increased in the fracture limb hind paw skin, sciatic nerve, and corresponding lumbar cord, peaking at 12 to 18 weeks after fracture and then declining. Immunohistochemistry localized postfracture IgM antibody binding to antigens in the fracture limb hind paw dermal cell nuclei. We postulate that fracture induces expression of neoantigens in the fracture limb skin, sciatic nerve, and cord, which trigger B cells to secret IgM antibodies that bind those antigens and initiate a pronociceptive antibody response. Autoimmunity plays a key role in the progression of nociceptive and vascular changes in the mouse fracture model and potentially contributes to the CRPS disease process.

Bisphosphonates Inhibit Pain, Bone Loss, and Inflammation in a Rat Tibia Fracture Model of Complex Regional Pain Syndrome.

BACKGROUND: Bisphosphonates are used to prevent the bone loss and fractures associated with osteoporosis, bone metastases, multiple myeloma, and osteogenesis deformans. Distal limb fractures cause regional bone loss with cutaneous inflammation and pain in the injured limb that can develop into complex regional pain syndrome (CRPS). Clinical trials have reported that antiresorptive bisphosphonates can prevent fracture-induced bone loss, inhibit serum inflammatory cytokine levels, and alleviate CRPS pain. Previously, we observed that the inhibition of inflammatory cytokines or adaptive immune responses attenuated the development of pain behavior in a rat fracture model of CRPS, and we hypothesized that bisphosphonates could prevent pain behavior, trabecular bone loss, postfracture cutaneous cytokine upregulation, and adaptive immune responses in this CRPS model. METHODS: Rats underwent tibia fracture and cast immobilization for 4 weeks and were chronically administered either subcutaneously perfused alendronate or oral zoledronate. Behavioral measurements included hindpaw von Frey allodynia, unweighting, warmth, and edema. Bone microarchitecture was measured by microcomputed tomography, and bone cellular activity was evaluated by static and dynamic histomorphometry. Spinal cord Fos immunostaining was performed, and skin cytokine (tumor necrosis factor, interleukin [IL]-1, IL-6) and nerve growth factor (NGF) levels were determined by enzyme immunoassay. Skin and sciatic nerve immunoglobulin levels were determined by enzyme immunoassay. RESULTS: Rats with tibia fractures developed hindpaw allodynia, unweighting, warmth, and edema, increased spinal Fos expression and trabecular bone loss in the lumbar vertebra and bilateral distal femurs as measured by microcomputed tomography, increased trabecular bone resorption and osteoclast surface with decreased bone formation rates, increased cutaneous inflammatory cytokine and NGF expression, and elevated immunocomplex deposition in skin and nerve. Alendronate (60 µg/kg/d subcutaneously [s.c.]) or zoledronate (3 mg/kg/d orally) treatment for 28 days, started at the time of fracture, completely inhibited the development of hindpaw allodynia and reduced hindpaw unweighting by 44% ± 13% and 58% ± 5%, respectively. Orally administered zoledronate (3 mg/kg/d for 21 days) treatment also completely reversed established allodynia and unweighting when started at 4 weeks postfracture. Histomorphometric and microcomputed tomography analysis demonstrated that both the 3 and 60 µg/kg/d alendronate treatments reversed trabecular bone loss (an 88% ± 25% and 188% ± 39% increase in the ipsilateral distal femur BV/TV, respectively) and blocked the increase in osteoclast numbers and erosion surface observed in bilateral distal femurs and in L5 vertebra of the fracture rats. Alendronate treatment inhibited fracture-induced increases in hindpaw inflammatory mediators, reducing postfracture levels of tumor necrosis factor by 43% ± 9%, IL-1 by 60% ± 9%, IL-6 by 56% ± 14%, and NGF by 37% ± 14%, but had no effect on increased spinal cord Fos expression, or skin and sciatic nerve immunocomplex deposition. CONCLUSIONS: Collectively, these results indicate that bisphosphonate therapy inhibits pain, osteoclast activation, trabecular bone loss, and cutaneous inflammation in the rat fracture model of CRPS, data supporting the hypothesis that bisphosphonate therapy can provide effective multimodal treatment for CRPS.

Acute versus chronic phase mechanisms in a rat model of CRPS.

BACKGROUND: Tibia fracture followed by cast immobilization in rats evokes nociceptive, vascular, epidermal, and bone changes resembling complex regional pain syndrome (CRPS). In most cases, CRPS has three stages. Over time, this acute picture, allodynia, warmth, and edema observed at 4 weeks, gives way to a cold, dystrophic but still painful limb. In the acute phase (at 4 weeks post fracture), cutaneous immunological and NK1-receptor signaling mechanisms underlying CRPS have been discovered; however, the mechanisms responsible for the chronic phase are still unknown. The purpose of this study is to understand the mechanisms responsible for the chronic phases of CRPS (at 16 weeks post fracture) at both the peripheral and central levels. METHODS: We used rat tibial fracture/cast immobilization model of CRPS to study molecular, vascular, and nociceptive changes at 4 and 16 weeks post fracture. Immunoassays and Western blotting were carried out to monitor changes in inflammatory response and NK1-receptor signaling in the skin and spinal cord. Skin temperature and thickness were measured to elucidate vascular changes, whereas von Frey testing and unweighting were carried out to study nociceptive changes. All data were analyzed by one-way analysis of variance (ANOVA) followed by Neuman-Keuls multiple comparison test to compare among all cohorts. RESULTS: In the acute phase (at 4 weeks post fracture), hindpaw allodynia, unweighting, warmth, edema, and/or epidermal thickening were observed among 90 % fracture rats, though by 16 weeks (chronic phase), only the nociceptive changes persisted. The expression of the neuropeptide signaling molecule substance P (SP), NK1 receptor, inflammatory mediators TNFα, IL-1ß, and IL-6 and nerve growth factor (NGF) were elevated at 4 weeks in sciatic nerve and/or skin, returning to normal levels by 16 weeks post fracture. The systemic administration of a peripherally restricted IL-1 receptor antagonist (anakinra) or of anti-NGF inhibited nociceptive behaviors at 4 weeks but not 16 weeks. However, spinal levels of NK1 receptor, TNFα, IL-1ß, and NGF were elevated at 4 and 16 weeks, and intrathecal injection of an NK1-receptor antagonist (LY303870), anakinra, or anti-NGF each reduced nociceptive behaviors at both 4 and 16 weeks. CONCLUSIONS: These results demonstrate that tibia fracture and immobilization cause peripheral changes in neuropeptide signaling and inflammatory mediator production acutely, but central spinal changes may be more important for the persistent nociceptive changes in this CRPS model.

Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization.

Tibia fracture induces exaggerated substance P (SP) and calcitonin gene-related peptide (CGRP) signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome. Inflammatory changes in the spinal cord contribute to nociceptive sensitization in a variety of animal pain models. This study tested the hypothesis that fracture-induced exaggerated neuropeptide signaling upregulates spinal inflammatory mediator expression, leading to postfracture hind limb nociceptive sensitization. At 4 weeks after performing tibia fracture and casting in rats, we measured hind limb allodynia, unweighting, warmth, edema, and spinal cord neuropeptide and inflammatory mediator content. The antinociceptive effects of intrathecally injected neuropeptide and inflammatory mediator receptor antagonists were evaluated in fracture rats. Transgenic fracture mice lacking SP or the CGRP RAMP1 receptor were used to determine the effects of neuropeptide signaling on postfracture pain behavior and spinal inflammatory mediator expression. Hind limb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF, IL-1, IL-6, CCL2, and nerve growth factor) were observed at 4 weeks after fracture in rats. Fracture-induced increases in spinal inflammatory mediators were not observed in fracture mice lacking SP or the CGRP receptor, and these mice had attenuated postfracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF, IL-1, IL-6, CCL2, or nerve growth factor each reduced pain behaviors in the fracture rats. Collectively, these data support the hypothesis that facilitated spinal neuropeptide signaling upregulates the expression of spinal inflammatory mediators contributing to nociceptive sensitization in a rodent fracture model of complex regional pain syndrome.

Immobilization contributes to exaggerated neuropeptide signaling, inflammatory changes, and nociceptive sensitization after fracture in rats.

UNLABELLED: A tibia fracture cast immobilized for 4 weeks can induce exaggerated substance P and calcitonin gene-related peptide signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome (CRPS). Four weeks of hind limb cast immobilization can also induce nociceptive and vascular changes resembling CRPS. To test our hypothesis that immobilization alone could cause exaggerated neuropeptide signaling and inflammatory changes, we tested 5 cohorts of rats: 1) controls; 2) tibia fracture and hind limb casted; 3) hind limb casted, no fracture; 4) tibia fracture with intramedullary pinning, no cast; and 5) tibia fracture with intramedullary pinning and hind limb casting. After 4 weeks, the casts were removed and hind limb allodynia, unweighting, warmth, edema, sciatic nerve neuropeptide content, cutaneous and spinal cord inflammatory mediator levels, and spinal c-Fos activation were measured. After fracture with casting, there was allodynia, unweighting, warmth, edema, increased sciatic nerve substance P and calcitonin gene-related peptide, increased skin neurokinin 1 receptors and keratinocyte proliferation, increased inflammatory mediator expression in the hind paw skin (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, nerve growth factor) and cord (IL-1ß, nerve growth factor), and increased spinal c-Fos activation. These same changes were observed after cast immobilization alone, except that spinal IL-1ß levels were not increased. Treating cast-only rats with a neurokinin 1 receptor antagonist inhibited development of nociceptive and inflammatory changes. Four weeks after fracture with pinning, all nociceptive and vascular changes had resolved and there were no increases in neuropeptide signaling or inflammatory mediator expression. PERSPECTIVE: Collectively, these data indicate that immobilization alone increased neuropeptide signaling and caused nociceptive and inflammatory changes similar to those observed after tibia fracture and casting, and that early mobilization after fracture with pinning inhibited these changes. Early limb mobilization after fracture may prevent the development of CRPS.

Epidermal adrenergic signaling contributes to inflammation and pain sensitization in a rat model of complex regional pain syndrome.

In many patients, the sympathetic nervous system supports pain and other features of complex regional pain syndrome (CRPS). Accumulating evidence suggests that interleukin (IL)-6 also plays a role in CRPS, and that catecholamines stimulate production of IL-6 in several tissues. We hypothesized that norepinephrine acting through specific adrenergic receptors expressed on keratinocytes stimulates the production of IL-6 and leads to nociceptive sensitization in a rat tibial fracture/cast model of CRPS. Our approach involved catecholamine depletion using 6-hydroxydopamine or, alternatively, guanethidine, to explore sympathetic contributions. Both agents substantially reduced nociceptive sensitization and selectively reduced the production of IL-6 in skin. Antagonism of IL-6 signaling using TB-2-081 also reduced sensitization in this model. Experiments using a rat keratinocyte cell line demonstrated relatively high levels of ß2-adrenergic receptor (ß2-AR) expression. Stimulation of this receptor greatly enhanced IL-6 expression when compared to the expression of IL-1ß, tumor necrosis factor (TNF)-α, or nerve growth factor. Stimulation of the cells also promoted phosphorylation of the mitogen-activated protein kinases P38, extracellular signal-regulated kinase, and c-Jun amino-terminal kinase. Based on these in vitro results, we returned to animal testing and observed that the selective ß2-AR antagonist butoxamine reduced nociceptive sensitization in the CRPS model, and that local injection of the selective ß2-AR agonist terbutaline resulted in mechanical allodynia and the production of IL-6 in the cells of the skin. No increases in IL-1ß, TNF-α, or nerve growth factor levels were seen, however. These data suggest that in CRPS, norepinephrine released from sympathetic nerve terminals stimulates ß2-ARs expressed on epidermal keratinocytes, resulting in local IL-6 production, and ultimately, pain sensitization.

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

BACKGROUND: Distal 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. RESULTS: Wildtype 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-1ß, 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. CONCLUSIONS: In 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 1ß (IL-1ß), interleukin 6 (IL-6), and nerve growth factor-ß (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.

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

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-1beta (IL-1beta) 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-1beta 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-1beta 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-1beta were co-expressed in keratinocytes, and the number of NALP1, caspase-1, and IL-1beta 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-1beta 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.

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

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.

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.

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. METHODS: The distal tibia was fractured and the hindlimb casted for 4 weeks. The rats were given drinking water with or without the cytokine inhibitor pentoxifylline (PTX) starting the day before fracture and continuing for 4 weeks, after which time the cast was removed and multiple assays were performed in the hindpaw. PCR and immunoassays were used to evaluate changes in cytokine expression. Bilateral hindpaw thickness, temperature, and nociceptive thresholds were determined, and bone microarchitecture was measured by microcomputed tomography (microCT). RESULTS: Tibia fracture chronically up-regulated TNFalpha, IL-1beta and IL-6 mRNA and protein levels in hindpaw skin and PTX treatment significantly reduced the mRNA expression and cytokine protein levels for all these cytokines. PTX inhibited the nociceptive sensitization and some vascular changes, but had insignificant effects on most of the bone-related parameters measured in these studies. Immunostaining of hindpaw skin was negative for immunocyte infiltration at 4 weeks post-fracture. CONCLUSIONS: These results suggest that pro-inflammatory cytokines contribute to the nociceptive and vascular sequelae of fracture and that PTX treatment can reverse these CRPS-like changes.

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

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 (microCT). 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.