Substance P Signaling Controls Mast Cell Activation, Degranulation, and Nociceptive Sensitization in a Rat Fracture Model of Complex Regional Pain Syndrome

• Skin dialysis studies in patients with complex regional pain syndrome (CRPS) are consistent with mast cell accumulation and degranulation, as well as substance P (SP) dysregulation

• In rats with tibia fracture and casting, a model of CRPS, mast cells accumulated around peptidergic nerve endings and degranulated with nerve stimulation in a manner consistent with SP release and its action on neurokinin-1 receptors

COMPLEX regional pain syndrome (CRPS) is a painful, chronic, and often disabling condition affecting the extremities. Type I CRPS, which does not involve primary nerve injury, is a frequent sequela of fractures of the distal tibia1and radius.2Recently, we developed a CRPS model in rats involving tibial fracture and cast immobilization that exhibits chronic unilateral hind limb warmth, edema, facilitated spontaneous protein extravasation, allodynia, unweighting, and periarticular osteoporosis.3This constellation of postfracture changes closely resembles the clinical presentation of acute or “warm” CRPS.4In this model, we observed the up-regulation of inflammatory cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor-α (TNF-α), and nerve growth factor in the epidermal keratinocytes of hind paw skin, and we demonstrated that inhibition of cytokine and nerve growth factor signaling during cast immobilization prevents allodynia and attenuates unweighting.5,–,10We also demonstrated that the neuropeptide substance P (SP) can initiate an interleukin-1β response in skin acting through neurokinin-1 (NK-1) receptors, which are themselves up-regulated in skin after fracture and immobilization.6,11Although epidermal keratinocytes have received the most attention in investigations involving neurocutaneous signaling, they are not the only cells in skin expressing NK-1 receptors, or the only cells capable of producing nociceptive mediators.

Cutaneous mast cells are a type of innate immune cell that resides in the dermis. They are characterized by abundant secretory granules that contain numerous preformed inflammatory mediators. They are intimately associated with cutaneous sensory nerves that can control degranulation.12,–,15When activated during tissue injury, mast cells are capable of releasing histamine along with various inflammatory mediators including cytokines, prostaglandin D², proteases, and other substances into the skin16that promote plasma protein leakage, vasodilation, and pain, characteristic of neurogenic inflammation. Making matters more complex, histamine has been shown to act through H1, H3, and H4 receptors in skin to cause pain and nociceptive sensitization in various models.17,–,19Mast cells are also major cellular participants in the development of chronic inflammatory skin diseases such as psoriasis, atopic dermatitis, and palmoplantar pustulosis.14,16,20–21The morphologic contacts between neurofilament-positive sensory nerves and tryptase-positive mast cells are more numerous in these skin diseases than in normal skin,14,20,–,22suggesting that the interaction between sensory nerves and mast cells plays a role in the pathogenesis of these diseases.

It has been shown that patients with CRPS have increased tryptase in the skin of the affected extremity, indicating increased mast cell activation and degranulation,23and it is well known that cutaneous mast cells express NK-1 receptor.24Based on these data and our observations concerning the increase of SP and NK-1 protein in the injured hind limb after fracture, we hypothesized that mast cell inward migration, activation, and degranulation may occur upon release of SP in the rat tibia fracture model of CRPS, and that NK-1-mediated mast cell degranulation can cause nociceptive sensitization. The demonstration of such a pathway would be novel to our understanding of the pathogenesis of CRPS, and would further support the role of neurocutaneous signaling in this condition.

These experiments were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA) and followed the animal subjects guidelines of the International Association for the Study of Pain.25Adult (9-month-old) male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) were used in all experiments. The animals were housed individually in isolator cages with solid floors covered with 3 cm of soft bedding and were given food and water ad libitum . During the experimental period the animals were fed Lab Diet 5012 (PMI Nutrition Institute, Richmond, IN), which contains 1.0% calcium, 0.5% phosphorus, and 3.3 IU/g vitamin D³, and were kept under standard conditions with a 12-h light-dark cycle.

Tibia fracture was performed under 2–4% isoflurane to maintain surgical anesthesia as we have previously described.3The right hind limb was wrapped in a stockinet (2.5 cm wide) and the distal tibia was fractured using pliers with an adjustable stop (Visegrip, Petersen Manufacturing, Dewitt, NE) that had been modified with a three-point jaw. The hind limb was wrapped in casting tape (δ-Lite, Johnson & Johnson, New Brunswick, NJ) so the hip, knee, and ankle were flexed. The cast extended from the metatarsals of the hind paw up to a spica formed around the abdomen. The cast over the paw was applied only to the plantar surface; a window was left open over the dorsum of the paw and ankle to prevent constriction when postfracture edema developed. To prevent the animals from chewing at their casts, the cast material was wrapped in galvanized wire mesh. The rats were given subcutaneous saline and buprenorphine immediately after procedure (0.03 mg/kg) and on the next day after fracture for postoperative hydration and analgesia. At 4 weeks the rats were anesthetized with isoflurane and the cast removed with a vibrating cast saw. All rats used in this study had union at the fracture site after 4 weeks of casting.

Animals were euthanized and perfusion performed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, via  the ascending aorta; the dorsal hind paw skin including subdermal layers was removed and postfixed in 4% paraformaldehyde for 2 h, then the tissues were treated with 30% sucrose in PBS at 4°C before embedding in optimum cutting temperature embedding compound (Sakura Finetek USA, Torrance, CA). After embedding, 10-μm-thick slices were made using a cryostat, mounted onto Superfrost microscope slides (Fisher Scientific, USA, Pittsburgh, PA), and stored at −70°C until use in histologic analysis and immunofluorescence confocal microscopy.

For histologic analysis of dermal mast cells, the sections were stained with toluidine blue and eosin.26After staining, randomly selected sections were examined under light microscopy. Based on the degree of degranulation and metachromasia, it is possible to identify three types of mast cells,27each representing a different stage in mast cell degranulation: (1) intact mast cells show a deep blue (orthochromatic) staining; (2) cells from which some granules have been extruded, but where the cell outline is largely intact and in which the granules demonstrate purple-red metachromasia; and (3) cells in which degranulation is more extensive and widespread in which the granules show metachromasia. A mast cell was considered to be degranulating if one or more extruded granules were visible adjacent to the cell. Because there is an element of subjectivity in distinguishing between types 2 and 3, the mast cells were divided into only two types, the intact and degranulating, encompassing types 2 and 3. For each skin sample, the total, intact, and degranulating mast cell numbers per 10 high- power fields (magnification × 400) were counted in the upper dermis. Each high-power field had an area of 10,000 um²and the total area examined per specimen was 100,000 μm².

Selective mast cell markers and immunofluorescence confocal microscopy were used to determine dermal mast cell activation and NK-1 receptor protein expression in mast cells. To assess mast cell proximity to cutaneous sensory nerves in the rat CRPS model, sections of hind paw skin were immunolabeled for a mast cell marker and costained for either SP or calcitonin gene-related peptide (CGRP). Briefly, frozen sections were permeabilized and blocked with PBS containing 10% donkey serum and 0.3% Triton X-100 before primary antibody incubation. Sections were incubated with primary antibody diluted in PBS containing 2% serum at 4°C overnight. After washing in PBS, the sections were incubated with fluorophore-conjugated secondary antibody. For double-labeling experiments, primary antibody from a different species against the second antigen was applied to the sections and visualized using an alternative fluorophore-conjugated secondary antibody. After three washes, the sections were mounted with antifade mounting medium (Invitrogen, Eugene, OR). Images were visualized using a confocal microscope (Zeiss LSM510 Upright 2 photon; Carl Zeiss, Jena, Germany). The primary antibodies used were rabbit antirat lysosome-associated membrane glycoprotein 1 (LAMP1), 1:1000 (LifeSpan Biosciences, Seattle, WA), fluorescein isothiocyanate-labeled rabbit antirat mast cell, 1:400 (Cedarlane Laboratories, Burlington, NC), rabbit antirat NK-1 receptor, 1:8000 (Sigma–Aldrich, St. Louis, MO), monoclonal mouse antirat keratin, 1:50 (clone AE1/AE3, Thermo Fisher Scientific, Fremont, CA), rabbit antirat SP, 1:6000 (Peninsula Laboratories Inc., San Carlos, CA), and rabbit antirat CGRP, 1:8000 (Sigma–Aldrich) were used. Double and triple labeling immunofluorescence was performed with donkey antimouse immunoglobulin G (1:500) conjugated with Dylight 549, donkey antirabbit immunoglobulin G (1:500) conjugated with Dylight 488, and donkey antirabbit immunoglobulin G (1:500) conjugated with Dylight 649 secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Control experiments included incubation of slices in primary and secondary antibody-free solutions, both of which led to low-intensity nonspecific staining patterns in preliminary experiments (data not shown). Quantitative studies were based on four or more replicates. Mast cells were counted per high-power field (400×) in the dermis of fracture and control rats. The numbers of mast cells in close proximity to nerve fibers (defined as ⩽2 μm) were counted separately. All slides were coded and counted under blinded conditions.

To test the hypothesis that SP signaling might regulate mast cell activity in the CRPS model, rats in the fracture group were treated with either an NK-1 receptor antagonist LY303870 (Eli Lilly Co., Indianapolis, IN) at a dose of 20 mg/kg/day intraperitoneally or saline for 8 days before euthanization. The dose was chosen on the basis of our previous studies.28We also tested the local effects of intraplantar SP injection in control rats on mast cell degranulation. The intact animals were treated with either SP (Sigma–Aldrich) at a dose of 25 μg/50 μl intraplantar or with saline. The dose was chosen on the basis of preliminary studies. The animals were euthanized and dorsal hind paw skin was harvested at 1, 3, and 6 h postinjection for histologic analysis of mast cell degranulation as mentioned previously.

To test the hypothesis that mast cells mediate the nociceptive and vascular changes observed after tibia fracture in rats, a mast cell degranulator, secretagogue compound 48/80 (Sigma–Aldrich), was administered intraperitoneally. For acute experiments, 400 μg 48/80 in sterile saline vehicle (0.9% NaCl) or vehicle alone was administered. Second, we tested the effects of 48/80 mediated mast cell depletion on nociceptive and vascular changes using escalating doses of 48/80: 50 μg (intraperitoneally) on the first day, 120 μg on the second day, 250 μg on the third day, and two injections of 400 μg on the fourth day (a total of 1.22 mg), chosen on the basis of our preliminary studies and the reports of others.29–30Control rats received vehicle alone. Additional animals were treated with 48/80 or saline before immunohistochemical analysis.

To assess if histamine supported warmth, edema, allodynia, and unweighting in the CRPS model, fracture rats were treated with either a highly selective H1 histamine receptor blocker, cetirizine (Sigma–Aldrich), at a dose of 5 mg/kg intraperitoneally, or saline vehicle.

We evaluated if electrical stimulation (ES) of sciatic nerves could induce mast cell degranulation and nociceptive sensitization in hind limb skin and if ES-caused mast cell degranulation is mediated by neuropeptide SP. Seventeen normal rats were divided into three cohorts: the first cohort of rats (n = 5) that served as control was sham-operated (sciatic nerve exposure without ES). The second cohort (n = 6) was subjected to 30 min of sciatic nerve ES (5 Hz, 0.5 ms pulse duration, 10 mA) under isoflurane anesthesia as described previously.11The third cohort (n = 6) was treated with LY303870 (intraperitoneally, 30 mg–40 mg/kg) 60 min before the ES. Nociceptive testing was performed at baseline and at 3 h after ES as described in Section 2.6. The animals were euthanized and perfusion performed with 4% paraformaldehyde in PBS, pH 7.4, via  the ascending aorta, at 3 h after ES chosen on the basis of preliminary studies; the dorsal hind paw skin including subdermal layers was harvested to assess mast cell degranulation by histologic analysis and immunofluorescence confocal microscopy as described previously.

To measure mechanical allodynia in the rats, an up-down von Frey testing paradigm was used as we have previously described.3,31 

An incapacitance device (IITC Inc. Life Science, Woodland Hills, CA) was used to measure hind paw unweighting. The rats were manually held in a vertical position over the apparatus with the hind paws resting on separate metal scale plates, and the entire weight of the rat was supported on the hind paws. The duration of each measurement was 6 s and 10 consecutive measurements were taken at 60-s intervals. Eight readings (excluding the highest and lowest 1 s) were averaged to calculate the bilateral hind paw weight bearing values.3,31 

A laser sensor technique was used to determine the dorsal-ventral thickness of the hind paw, as we have previously described.3,28,31 

For these experiments room temperature was maintained at 23°C and humidity ranged between 25% and 45%. The temperature of the hind paw was measured using a fine wire thermocouple (Omega, Stamford, CT) applied to the paw skin, as previously described.3,28,31 

Statistical analysis was performed using a two-way analysis of variance (ANOVA) followed by Bonferroni post hoc  testing to compare the time course between the control cohort (fracture only) and the fracture rat cohorts that were injected with mast cell degranulator 48/80 or histamine receptor 1 antagonist cetirizine treated fracture rats. One-way ANOVA was used followed by post hoc  Newman-Keuls multiple-comparison testing to compare means of three samples. For simple comparisons of two means, unpaired Student t  test was performed. All data are presented as the mean ± standard error of the mean, and differences are considered significant at a P  value less than 0.05 (Prism 5, GraphPad Software, San Diego, CA).

We first assessed mast cell numbers and degranulation in hind paw skin harvested from cast immobilization, fracture with cast, and control rats. Skin sections were incubated with an antibody directed against the rat keratin Pan-Ab1 antigen and costained with a selective mast cell marker, which recognizes both activated and inactivated mast cells. We observed only a few mast cells in the superficial dermis in control rats (fig. 1A), and a small increase in mast cell number and degranulation in the upper dermis in rats with cast (fig. 1B), whereas the number of detectable mast cells and degranulation were dramatically increased in the upper dermis adjacent to basal membrane 4 weeks postfracture (fig. 1C). Figure 1D illustrates a sevenfold and fourfold increase in positive mast cells in the skin of the fractured hind limb compared with control and cast animals, respectively, suggesting that the changes in mast cells is largely produced by the distal tibia fracture.

Next, mast cell activation in dermis of postfracture rats was confirmed by immunostaining of lysosome-associated membrane glycoprotein 1 (LAMP-1, i.e. , CD107a), a selective active mast cell marker.32No LAMP-1 positive cells were observed in normal hind paw skin (fig. 2A), whereas at 4 weeks postfracture large numbers of LAMP-1 immunoreactive cells were present in the dermis of the hind paw ipsilateral to fracture (fig. 2B), but only a few LAMP-1 immunostained cells were observed in the contralateral hind paw (fig. 2C), indicating that long-term activation of dermal mast cells was restricted to the injured limb.

Finally, dermal mast cell degranulation after fracture was confirmed using histologic analysis of the hind limb sections with toluidine blue and eosin staining. Figure 3A shows representative light micrographs of histologic staining for mast cells from control rats and from the skin of hind paws ipsilateral to fracture at 4 weeks postinjury. In normal rats, dermal mast cells were intact with dark blue staining (fig. 3A), whereas degranulating mast cells appearing purple-red were observed 4 weeks postfracture (fig. 3, B and C). There was a 390% increase in mast cell number (fig. 3D) and 250% increase in the percentage of degranulated mast cells (fig. 3E) in the upper dermis of fractured hind limb compared with control rats. Degranulation was prominent in the region of the dermal-epidermal boundary.

We next hypothesized that increased SP signaling after fracture causes mast cell activation and degranulation in the skin. To test the hypothesis, fracture rats were treated with either an NK-1 receptor antagonist (LY303870) or saline for 8 days before cast removal. Figure 4A, lower panel, shows representative confocal immunofluorescence microscopy results for mast cells in dorsal hind paw skin from LY303870 treated fracture animals, and fig.4B presents quantification of mast cells in the hind paw dermis. These results demonstrate that treatment after fracture with LY303870 completely reversed the fracture-induced increases in mast cell number.

We also looked at the change of NK-1 receptor expression in hind paw skin in untreated and in LY303870 treated fracture rats. Figure 5demonstrates NK1 receptor expression in dermal mast cells and epidermal keratinocytes, with a clear increase in NK1 receptor expression in both types of cells at 4 weeks after fracture (fig. 5, A and B), whereas LY 303870 treatment prevented these fracture-induced changes (fig. 5C). We previously had observed a similar increase in epidermal keratinocyte NK-1 expression in the rat fracture model.11 

Functional peripheral neuron-mast cell communication in skin has been inferred based on increased nerve fiber-mast cell proximity in other disease models, and we examined fracture rat skin for evidence of such changes. Sections of hind paw skin from control rats and 4 weeks postfracture rats were stained with anti-SP, anti-CGRP and antimast cell antibodies, to identify peptidergic nerves and mast cells, respectively. As shown in figure 6A and B, we observed close associations between mast cells and SP and CGRP positive nerves in hind paw skin at 4 weeks postfracture. When expressed as the percentage of dermal mast cells that occurred within 2 μm of the nearest nerve fiber, we found that the percentage of mast cells in close proximity with SP and/or CGRP nerve fibers was significantly increased in the hind paw skin at 4 weeks postfracture (fig. 6C).

The findings previously are suggestive of functional communication between neuropeptide containing nerve fibers and mast cells. To further evaluate this view, we investigated whether ES of sciatic nerve could induce mast cell activation, degranulation, and nociceptive sensitization as well as whether an NK-1 receptor antagonist (LY303870) could block any possible ES-induced effects. Our data showed that antidromic ES of sciatic nerve induced mast cell activation (fig. 7A), degranulation (fig. 7B by toluidine blue-eosin staining and fig. 7C by a mast cell marker), and nociceptive sensitization (fig. 7, E and F). There was a 210% increase in the percentage of degranulated mast cells in the dermis at 3 h after electrical stimulation compared with controls (fig. 7D). The antagonist LY 303870 blocked these changes (fig. 7).

Next, we examined the local effects of intraplantar SP injection in control rats without fracture on mast cell degranulation. As shown in figure 8, intraplantar SP injection induced extensive mast cell degranulation in the plantar hind paw skin at 1, 3, and 6 h postinjection.

Because mast cell degranulation releases a variety of inflammatory mediators such as histamine, cytokines, prostaglandin D², and tryptase into the skin, and ES of sciatic nerve induces mast cell degraulation and hind limb nociceptive changes, we hypothesized that mast cell degranulation can exacerbate nociceptive sensitization in the fracture CRPS model. To test this hypothesis, a mast cell degranulator and secretagogue compound 48/80 was administered systemically at 4 weeks postfracture. We observed that acute administration of 48/80 (400 μg/rat, intraperitoneally) caused additional mast cell degranulation (data not shown) and enhanced postfracture allodynia (fig. 9A), but had no additional effects on weightbearing (fig. 9B) or warmth (fig. 9C) at 4 weeks postfracture compared with vehicle- treated fracture rats. There was a trend toward elevation of hind paw thickness at 3 h postinjection of 48/80 (fig. 9D). The acute administration of 48/80 had no effects on paws contralateral to fracture.

To further evaluate our hypothesis that mast cells mediate the nociceptive and vascular changes observed after tibia fracture in rats, ascending doses of compound 48/80 were administered systemically for 4 days before testing and skin harvest at 4 weeks postfracture. Figure 10presents the effects of chronic 48/80 treatment (1.22 mg/rat, intraperitoneally total) on mast cell degranulation, nociceptive and vascular changes at 4 weeks postfracture. Fracture caused a fourfold increase in mast cell numbers, and chronic 48/80 treatment reversed this increase (fig. 10, A and B). Furthermore, chronic 48/80 treatment partially reversed the hind paw allodynia and unweighting that developed in the fracture rats (fig. 10, C and D), but no effects were observed on hind paw warmth (fig. 10E). Collectively, these results strongly support our hypothesis that mast cells play a role in the nociceptive changes observed after tibia fracture.

Histamine is a commonly studied preformed mediator in mast cell secretory granules that is released upon activation, e.g. , by SP or an allergen, and it is able to induce nociceptive responses via  histamine receptors, most notably of the H1 subtype.33,–,37We therefore tested if histamine acting through H1 receptors is required for the warmth, edema, allodynia, and unweighting in the CRPS model. Fracture rats were treated with either a highly selective H1 histamine receptor blocker, cetirizine at a dose of 5 and 10 mg/kg intraperitoneally, or saline. Unweighting, temperature, edema, and mechanical thresholds were tested in the hind paws at 1, 3, 6 and 24 h after cetirizine injection. Figure 11demonstrates that acute administration of cetirizine failed to block nociceptive and vascular changes (data not shown) in the CRPS model.

Patients with CRPS have increased tryptase in the skin of the affected extremity, indicating increased mast cell accumulation and degranulation.23Mast cells release a number of inflammatory mediators such as histamine, cytokines, prostaglandin D², and tryptase into the skin, and we hypothesized that mast cell degranulation contributes to nociceptive sensitization in a rat tibia fracture model of CRPS. Furthermore, after tibia fracture SP signaling in the injured hind limb skin is up-regulated, and we observed previously that treating fracture rats with an NK-1 receptor antagonist (LY303870) attenuates nociceptive sensitization in the hind paw.3It is well known that mast cells express NK-1 receptors and are capable of responding to SP.24We therefore pursued the hypothesis that increased SP signaling through NK-1 receptors after fracture causes mast cell accumulation, activation and degranulation in the skin resulting in nociceptive sensitization. Finally, we tested if H1 histamine receptors contribute to the nociceptive sensitization after tibia fracture.

In this study, using a polyclonal antimast cell antibody that recognizes both active and inactive mast cells, we showed that only a few mast cells were present in the superficial dermis of control unfractured rats, and only a small increase in mast cell number was observed in the upper dermis 4 weeks postcast in unfractured animals. However, the number of detectable mast cells was dramatically increased in the upper dermis in the combined fracture/cast animals, which most faithfully replicates the signs of CRPS in humans.3Using LAMP-1, an activated mast cell marker,32we demonstrated that LAMP-1 positive cells were absent in normal skin, whereas at 4 weeks postfracture there was a dramatic increase in LAMP-1 immunoreactive cells in the dermis, suggesting the activation of mast cells after fracture. Consistent with the LAMP-1 immunoreactivity were the direct histologic observations of degranulation using toluidine blue-eosin staining. Collectively, these data indicate that tibia fracture leads to both the accumulation and, perhaps more importantly, the activation of degranulation in hind paw skin. This finding is in agreement with the previous work of Huygen et al.  23in CRPS patients where enhanced tryptase concentrations were observed in the fluid from suction blisters made in the involved versus  uninvolved CRPS extremity. Mast cell activation has also been detected in different painful conditions such as wound healing,38nerve injury,37migraine headache,39–40interstitial cystitis,41chronic pancreatitis,42–43and inflammatory arthritis,44and in chronic inflammatory skin diseases such as psoriasis, atopic dermatitis, and palmoplantar pustulosis.14,16,20–21The aforementioned studies provided evidence for a range of mediators being responsible for the mast cell activation, e.g. , through endogenous proteins such as TNF, tryptase, complement component 5a, SP, and vasoactive intestinal polypeptide as well as by histamine release.

One key question is how fracture can cause cutaneous mast cell accumulation, activation, and degranulation. Although leukocyte-mediated inflammation is absent in CRPS- affected skin and joints, facilitated neurogenic signaling and inflammation has been demonstrated in several studies.45,–,47Similarly, after tibia fracture and immobilization in rats, increased SP signaling and NK-1 receptor expression are observed in sensory neurons and epidermal keratinocytes in the fractured limb. These changes lead to keratinocyte activation, proliferation, and overexpression of proinflammatory mediators.3,5,28Neuroimmune signaling involving the peripheral nervous system and mast calls is also well documented. For example, mast cells in close proximity to neurites are known to form “synapse-like” structures involving the cell adhesion molecule N -cadherin.48Cutaneous mast cell degranulation has been shown to rely on the integrity of such synapse-like structures formed with peptidergic neurons; disruption of these N -cadherin-containing structures by deletion of the gene coding for the membrane-type 5 matrix metalloproteinase alters mast cell degranulation and nociceptive sensitization.49 

The close association of sensory nerves and mast cells is implicated in several inflammatory skin diseases12,–,14and in wound healing.38We thus hypothesized that facilitated neurocutaneous signaling also contributes to the dermal mast cell activation and degranulation after fracture. Our data demonstrate that close associations between mast cells and neuropeptide nerve fibers were present in hind paw skin at 4 weeks postfracture, and the percentage of mast cells in close proximity with SP and/or CGRP nerve fibers was significantly increased in the hind paw skin after fracture. These findings are suggestive of functional communication between peptidergic neuron and mast cells; to support this view, we first detected that mast cells present in skin after fracture express NK-1 receptors, and that an NK-1 antagonist blocked the fracture-induced increase in mast cell degranulation. We then observed that ES of the sciatic nerve significantly degranulated dermal mast cells and induced nociceptive responses. However, the ES-induced mast cell degranulation and nociceptive response were blocked in rats pretreated with a NK-1 antagonist. Moreover, we showed that intraplantar SP injection induces mast cell degranulation. It should be noted that increased concentration of SP in the skin has been measured in the CRPS model as has augmented NK-1 receptor-dependent neurogenic edema.11Others have reported that ES of sciatic nerve can cause a significant increase in dermal mast cell degranulation and histamine release in rat hind paws.50–51However, this response was significantly blocked in rats pretreated in the neonatal period with capsaicin to reduce afferent neuropeptide signaling.50Thus, our observations that the number of mast cells increases and the proximity of the mast cells to neuropeptide expressing nerve fibers is likely functionally meaningful.

Pain and hyperalgesia are the most distressing symptoms in CRPS, which result, at least partially, from activation or sensitization of peripheral nociceptors.52Because of their large repertoire of inflammatory mediators such as histamine, cytokines, prostaglandins, and proteases,53we speculated that mast cell degranulation could cause nociceptive sensitization in the CRPS model. To address this hypothesis, we next administered a mast cell degranulator, compound 48/80 at 4 weeks postfracture. The acute administration of 48/80 caused mast cell degranulation and enhanced postfracture nociception, but nociceptive behavior was attenuated when mast cells were depleted after 4 days of 48/80 treatment. These observations strongly suggest that mast cell degranulation after fracture can induce sensitization, thus providing new evidence for the importance of neurocutaneous signaling in the development of CRPS. Previous studies suggest that increased mast cell number and degranulation are associated with the pathogenesis of chronic pain in inflammatory conditions,30,36after peripheral nerve injury,37and in several human diseases such as migraine headache,39–40interstitial cystitis,41and chronic pancreatitis.42–43 

We investigated the effects of cetirizine, a selective H1 histamine receptor blocker, on nociceptive and vascular changes in the CRPS model. Mast cell derived histamine may support sensitization in other pain models acting through the H1 receptor.33,–,37,54Surprisingly, our studies clearly show that acute administration of cetirizine failed to block nociceptive and vascular changes observed in the CRPS model. It should be noted that H1 blockade did not reduce the enhanced vascular leakage induced by the acute injection of 48/80 in a separate study.55We cannot rule out the possibility of histamine supporting sensitization via  other histamine receptors because recent reports suggest the H3 and H4 receptors may mediate nociception in skin.17–18 

Evidence from other groups suggests that mast cells may induce nociceptive sensitization through the action of nonhistamine granule contents. For example, nerve growth factor-β derived from mast cells sensitizes nociceptors and also increases expression of tryptase in mast cells.56–57We demonstrated previously that nerve growth factor is an active pain-related mediator in the rat fracture model.49SP via  activation of NK-1 receptors on mast cells increases expression of TNF,58–59which in turn sensitizes nociceptive terminals via  activation of the TNF receptors. Again, TNF-α supports nociceptive changes in the model used here.8Finally, mast cells may facilitate nociceptive sensitization by activating surrounding cells such as keratinocytes. There is growing evidence that keratinocytes express protease activated receptor 2 and histamine receptors, which can be activated by mast cell proteases and histamine, respectively,60–61thus enhancing the expression of inflammatory cytokines, matrix metalloproteinases, and antimicrobial peptides.60,62,–,64In this regard it is also notable that TRPA1 receptors are located in proximity to protease-activated receptor 2 receptors, and that activation of TRPA1 receptors by protease activated receptor 2 receptors may enhance the sensitivity of TRPA1 receptors during inflammation.65The observation of increased numbers of mast cells and apparent degranulation in the upper dermis in close proximity to the basal layer of the epidermis support this possible mechanism in this CRPS model.

In summary, these experiments indicate that facilitated sensory neuron-mast cell signaling after fracture can cause mast cell accumulation and degranulation into the skin of the injured limb and induce nociceptive sensitization. Future experiments are needed to determine which mast cell granule contents mediate pain and inflammation in this model. Given the diversity of mast cell granule contents, it seems possible that multiple parallel mechanisms are involved. An improved understanding of mast cell function in CRPS might lead to novel strategies for preventing CRPS in high-risk patients or reducing the severity of the condition once established.