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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Anesthesiology. 2010 Oct;113(4):945–956. doi: 10.1097/ALN.0b013e3181ee2f17

Caspase-1 Modulates Incisional Sensitization and Inflammation

De-Yong Liang 1,2, XiangQi Li 1,2, Wen-Wu Li 1,2, Dennis Fiorino 3, Yanli Qiao 2, Peyman Sahbaie 1,2, David C Yeomans 2, J David Clark 1,2,4
PMCID: PMC2945456  NIHMSID: NIHMS221453  PMID: 20823759

Abstract

Background

Surgical injury induces production and release of inflammatory mediators in the vicinity of the wound. They in turn trigger nociceptive signaling to produce hyperalgesia and pain. IL-1β plays a crucial role in this process. The mechanism regulating production of this cytokine after incision is, however, unknown. Caspase-1 is a key enzyme that cleaves pro-IL-1β to its active form. We hypothesized that caspase-1 is a crucial regulator of incisional IL-1β levels, nociceptive sensitization and inflammation.

Methods

These studies employed mouse hindpaw incisional model. Caspase-1 was blocked using the selective inhibitors Ac-YVAD-CMK and VRTXSD727. Nociceptive sensitization, edema and hindpaw warmth were followed in intact animals while caspase-1 activity, cytokine and PGE2 levels were assessed in homogenized skin. Confocal microscopy was used to detect the expression of caspase-1 near the wounds.

Results

Analysis of enzyme activity demonstrated that caspase-1 activity was significantly increased in peri-incisional skin. Pretreatment with Ac-YVAD-CMK significantly reduced mechanical allodynia and thermal hyperalgesia. Repeated administration of this inhibitor produced robust analgesia especially to mechanical stimulation. Administration of VRTXSD727 provided qualitatively similar results. Caspase-1 inhibition also reduced edema and the normally observed increase in paw warmth around the wound site. Correspondingly, caspase-1 inhibition significantly reduced IL-1β as well as MIP-1α, G-CSF and PGE2 levels near the wound. The expression of caspase-1 was primarily observed in keratinocytes in epidermal layer and in neutrophils deeper in the wounds.

Conclusions

The current study demonstrates that the inhibition of caspase-1 reduces postsurgical sensitization and inflammation likely through a caspase-1/IL-1β dependent mechanism.

Introduction

Postoperative pain is an expected consequence of most surgeries. Unfortunately, 30–40% of patient suffer moderate to severe pain during the postoperative period despite the aggressive use of available medications 1,2. One of the root causes of this pain involves the surgically induced production and release of a variety of inflammatory mediators constituting an “inflammatory soup” at the wound site. These mediators have known roles in wound healing and in fighting infection. However, proinflammatory cytokines also represent components of the inflammatory soup implicated in generating pain in postsurgical, inflammatory, musculoskeletal and perhaps neuropathic pain states 3–8. Specific to the tissue surrounding incisions, elevated levels of many cytokines have been reported in rodents and humans including Interleukin (IL)-1 β, IL-6, tumor necrosis factor α, granulocyte colony stimulating factor (G-CSF), macrophage-inflammatory protein 1α (MIP-1α), keratinocyte-derived cytokine and others5–7,9,10. The archetypical proinflammatory cytokine IL-1β has been implicated strongly in supporting pain during inflammation and after tissue injury5,6,9,11–13. Furthermore, administration of IL-1 receptor antagonist sharply reduces nociceptive sensitization surrounding incisions in rodents 9. Beyond its direct sensitizing effects, IL-1β induces the production of other proinflammatory cytokines and mediators linked to nociception including Tumor necrosis factor α, IL-6, cyclooxygenase-2, chemokines, Substance P and nerve growth factor 14–17. Very little is known, however, about the mechanisms supporting IL-1β production and release after incision.

Caspase-1 (also known as IL-1β-converting enzyme) is a cysteine protease that cleaves pro-IL-1β, pro-IL-18 and pro-IL-33 to form the mature active cytokines 18,19. Caspase-1 was the first member identified in the caspase family of cysteine proteases that now has 14 known members, 11 of which are expressed in human tissue 20. This family of proteases has critical roles in apoptosis and inflammation and includes inflammatory caspases (caspase-1, -4, -5, -11, and – 12), initiator caspases (-2, -8, -9, and -10) and executioner (apoptotic) caspases (caspase-3, -6, and -7). Caspase-1 is constitutively expressed in many tissues, and is highly inducible in macrophages, T cells, neutrophils and kerotinocytes. Caspase-1 inhibition has shown extraordinary promise in multiple disease models including ones for painful conditions. For example, blockade of caspase-1 activity reduces inflammation, neuropathic nociceptive sensitization and dynorphin-induced allodynia in rodent models 12,15,21,22. We hypothesized that wound area caspase-1 supports postincisional IL-1β production, nociceptive sensitization, edema, warmth, and the generation of inflammatory mediators downstream of IL-1β which also support nociception.

Materials and Methods

Animals

All experimental protocols were reviewed and approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, California) before the initiation of work. All protocols conform to the guidelines for the study of pain in awake animals as established by the International Association for the Study of Pain. Male C57BL/6 mice 8–9 weeks old were obtained from The Jackson Laboratory (JAX; Bar Harbor, ME). Mice were housed 4 per cage, and maintained on a 12-h light/dark cycle and an ambient temperature of 22 ± 1°C, with food and tap water available ad libitum.

Drug Administration

N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-CMK), a selective caspase-1 inhibitor, (Calbiochem, San Diego, CA) was freshly dissolved in dimethyl sulfoxide then diluted in 0.9% NaCl prior to use. For local intraplantar injection, Ac-YVAD-CMK was injected in a volume of 10µl 0.9% NaCl/2.5% dimethyl sulfoxide final concentration at 24 h postincision. Systemic administration involved the subcutaneous injection of 100µl NaCl of the same solution. In some experiments animals received the first treatment 30 min prior to incision, and the second treatment 24 h postincision. In other experiments, animals received the first treatment of this compound only 24 h after incision. VRTXSD727, a gift of Vertex Pharmaceuticals(San Diego, CA) was freshly dissolved in 25% cremophor for use, and was administered via oral gavage (p. o.) 10 ml/kg, twice daily. Oral administration of VRTXSD727 was performed 2 h prior to behavioral testing on each day.

Surgical Preparation

Paw incision in mice was performed as described previously in our lab 5–7. Briefly, mice were anesthetized with isoflurane delivered via a nose cone. After sterile preparation of the right hindpaw, a 0.5 cm longitudinal incision was made through skin and fascia of the plantar foot with a number 11 scalpel blade. The incision was started 0.2 cm from the proximal edge of the heel and extended distally. The underlying muscle was elevated with curved forceps, leaving the muscle origin and insertion intact. After wound hemostasis, the skin was opposed with a 6.0 nylon mattress suture and the wound was covered with antibiotic ointment. In some experiments, control mice without incision underwent a sham procedure that consisted of anesthesia, antiseptic preparation, and application of the antibiotic ointment without an incision.

Pain behaviors

Mechanical allodynia

Mechanical nociceptive thresholds were assayed using von Frey filaments according to the “up-down” algorithm described by Chaplan et al.23 as we described previously 24,25. Mice were placed on wire mesh platforms in clear cylindrical plastic enclosures of 10 cm diameter and 30 cm in height. After 20 min of acclimation, fibers of sequentially increasing or decreasing stiffness with beginning bending force of 0.2 gram were applied to the plantar surface of the right hindpaw adjacent to the incision, just distal to the first set of foot pads and left in place 5 s with enough force to slightly bend the fiber. Withdrawal of the hindpaw from the fiber was scored as a response. When no response was obtained, the next stiffer fiber in the series was applied to the same paw. If a response was obtained, a less stiff fiber was next applied. Testing proceeded in this manner until four fibers had been applied after the first one causing a withdrawal response allowing the estimation of the mechanical withdrawal threshold using a curve fitting algorithm 26.

Thermal Hyperalgesia

Paw withdrawal response latencies to noxious thermal stimulation were measured using the method of Hargreaves et al. 27 as we have modified for use with mice 24. In this assay, mice were placed on a temperature-controlled glass platform (29 °C) in a clear plastic enclosure similar to those described above. After 30 min of acclimation, a beam of focused light was directed towards the same area of the hindpaw as described for the von Frey assay. A 20-s cutoff was used to prevent tissue damage. In these experiments, the light beam intensity was adjusted to provide an approximate 10-s baseline latency in control mice. Three measurements were made per animal per test session separated by at least 1 min.

Hindpaw edema

Paw thickness from dorsal side to ventral side was measured by a laser sensor technique as previously described by Guo and et al.28, as modified for mice by Clark and et al. 5. Briefly, mice were first anesthetized by exposure to isoflurane. Each hindpaw was then held in turn against a flat surface, above which was affixed a laser device capable of triangulating thickness with a precision of 0.01 mm (model 4381 Precicura; Limab, Goteborg, Sweden). Paw thickness was measured over the third metatarsal at a point 1–2 mm distal to the most distal aspect of the incision. For each animal, three measurements were made of both the incised and nonincised hindpaws. The ratio of these thickness measurements was used to compare mice.

Hindpaw temperature

The temperature of the hind paw was measured using a fine wire thermocouple (Omega, Stamford, CT) applied to the paw skin previously described in rats by Guo et al28. Briefly, the investigator held the device using an insulating Styrofoam block. Three sites were tested twice each over the dorsum of the hindpaw; the space between the first and second metatarsals (medial), the second and third metatarsals (central), and the fourth and fifth metatarsals (lateral). After a site was tested in one hindpaw, the same site was immediately tested on the contralateral hindpaw. The six measurements for each hindpaw were averaged for the mean temperature.

Skin tissue harvest and protein isolation

Mice were euthanized immediately after behavioral measurement at the time points specified in the figures. The skin tissue surrounding incision with approximate 1.5mm margins was excised, and skin specimens were placed into phosphate-buffered saline containing a protease inhibitor cocktail (Roche Complete, Roche Diagnostics, Mannheim, Germany), and frozen at −8°C until analysis. For use these samples were first cut into small pieces with microscissors then disrupted using a Polytron Device (Brinkmann Instruments Inc, Westbury, NY). The samples were then centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was carefully pipetted into a fresh 1.5 ml tube, which was the material used for protein analysis. Protein concentration was evaluated with a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA).

Cytokine and prostaglandin E2 assays

Cytokines IL-1β, MIP-1α and G-CSF were analyzed by BioPlex mouse cytokine assay (Bio-Rad, Laboratories, Hercules, CA) as previously described 5–7. In brief, premixed beads (50 µl) coated with target capture antibodies were transferred to each well of the filter plate supplied with the assay kit and washed twice with Bio-Plex wash buffer. Premixed standards or samples (50 µl) were added to each well containing washed beads. The plate was shaken for 30 s and then incubated at room temperature for 30 min with low-speed shaking. After incubation and washing, premixed detection antibodies (50 µl) were added to each well. The incubation was terminated after shaking for 10 min at room temperature. After washing three times, the beads were resuspended in 125 µl of Bio-Plex assay buffer. Beads were read on the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software with 5PL curve fitting.

Prostaglandin E2 (PGE-2) level was estimated by enzyme-linked immunosorbent assay*** measurement of the PGE-2 metabolite. This assay was conducted according the kit manufacturer’s instructions, and used the provided standards (Cayman Chemical Co., Ann Arbor, MI). Briefly, samples were incubated in phosphate and carbonate buffer prepared with deionized water and incubated overnight at 37°C to convert intact PGE-2 and its intermediate metabolites to a stable PGE-2 metabolite. The concentration of PGE-2 metabolite in the samples was determined by using a specific enzyme-linked immunosorbent assay***. The metabolite assay was chosen since PGE-2 is rapidly metabolized in vivo and consequently does not accurately reflect endogenous prostaglandin production 29.

Caspase-1 activity assays

The activity of caspase-1 was determined by use of a fluorometric assay kit according to the manufacture’s protocol obtained from BioVision (BioVision, Mountain View, CA). Briefly, a volume of 50 µl of protein sample from skin tissue was added to 50 µl of 2x caspase-1 reaction buffer (containing 10 mM dithiothreitol) in a duplicate manner in a 96-well plate. Then, 1mM caspase-1 substrate, YVAD-AFC (5 µl) was added to each sample, followed by 2 h of incubation at 37 °C. The fluorescence levels were was measured in a microplate spectrofluorometer equipped with a 400 nm excitation filter and 505 nm emission filter (SpectraMax GEMINI, Molecular Devices, Sunnyvale, CA) reader. The fold-increase in caspase-1 activity was determined by use of a standard curve.

Immunohistochemical Analysis

We previously reported our methods for the immunohistochemical analysis of incised mouse paw skin 5,6. For these analyses mice were sacrificed using carbon dioxide asphyxiation, and was followed by intra cardiac perfusion of 20 ml 0.9% NaCl followed by 20 ml of 10% neutrally buffered formalin. Hind paws were then removed and incubated in 10% buffered formalin for 24 h. After overnight decalcification, tissue was processed for paraffin sectioning in automated fashion (Tissue Tek VIP, Miles Scientific, Naperville, IL). Following embedding, 6.0 µM slices were made then placed on slides and incubated for 20 min at 55°C to improve adherence. Paraffin was removed with graded xylenes then rehydrated in ethanol. Blocking of these sections took place overnight at 4°C in Tris-buffered saline containing 5% dry milk, followed by exposure to the primary antibodies against active-caspase-1, IL-1β or neurophils overnight at 4 °C in milk-Tris-buffered saline. The primary antibodies included polyclonal anti-IL-1β, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-cleaved (mature) caspase-1, 1:200 (Abcam Inc., Cambridge, MA) and anti-mouse neutrophil antibody, 1:5000 (ABD Serotec, Kidlington, Oxford, United Kingdom). For specificity of IL-1β antibody the preabsorption of the antibody with blocking peptide was conducted before adding to the section. Sections were then be rinsed and transferred to milk-Tris-buffered saline containing flourescein conjugated secondary antibodies against the primary antibodies, 1:300–1:500 (Jackson ImmunoResearch Laboratories, West Grove, PA) and incubated for 1 h. After washing, coverslips were applied. Confocal laser-scanning microscope was carried out using a Zeiss LSM/510 META microscope (Thornwood, NY). Images were stored on digital media. Control experiments included incubation of slices in primary and secondary antibody free-solutions, both of which lead to low intensity nonspecific staining patterns in preliminary experiments.

Statistical analysis

The results are expressed as mean ± SEM. The data of mechanical sensitivity, thermal sensitivity, edema, paw warmth, and cytokines were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test for multiple comparisons. For simple comparisons of two groups a two tailed Student’s t-testing was employed. P values less than 0.05 were considered significant (Prism 4, GraphPad Software, La Jolla, CA).

Results

Caspase-1 Activity After paw incision

Caspase-1 activity is a key step modulating IL-1β production. We first directly assessed this enzyme activity in the skin surrounding incisions in mice. Figure 1 shows that caspase-1 activity was significantly and transiently increased after paw incision.

Fig. 1.

Fig. 1

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Incision enhances caspase-1 activity after incision. The data is expressed as the fold-change relative to control. Values expressed as mean ± SEM, n = 6, *** p < 0.001 compared to nonincision group (t = 0 h).

Caspase-1 inhibition suppresses mechanical allodynia after paw incision

Mechanical allodynia is an important feature of human surgical wounds and is present in the rodent model of incisional pain for several days after incision 30,31. In our experiments mechanical allodynia reduced withdrawal thresholds for the entire 48-h period over which the mice were observed (F10, 165=35.35; P < 0.0001), as shown as figure 2A. Systemic administration of Ac-YVAD-CMK 30 min before incision reduced the allodynia with maximal effectiveness 1 to 2 h after incision declining thereafter (F20,165=5.44; P < 0.0001). A small but significant analgesic effect was observed even 24-h postincision. When inhibitor administration was repeated in these animals mechanical allodynia was completely reversed, an effect which persisted for another 24 h. Contralateral withdrawal thresholds were not altered by inhibitor administration (data not shown).

Fig. 2.

Fig. 2

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on mechanical sensitivity in paw incision model. A. Effect of two subcutaneous injections of Ac-YVAD-CMK on mechanical sensitivity. The first injection was pretreatment at 0.5 h before paw incision, the second application was made at 24 h after paw incision. B. Effect of Ac-YVAD-CMK on mechanical sensitivity. The subcutaneous injection was made at 24 h post-paw incision. C. Effect of Ac-YVAD-CMK local intraplantar injection on mechanical sensitivity 24 h after paw incision. Solid line was Ac-YVAD-CMK group, dash line was control group. Values are displayed as mean ± SEM, n = 6, * p < 0.05 or *** p < 0.001 Nonincision group compared to the control group; ## p < 0.01 or ### p < 0.001 in comparison of Ac-YVAD-CMK group with the control group

To further explore the unexpected efficacy of repeated inhibitor administration, we conducted a separate experiment where Ac-YVAD-CMK was administered to incised animals first at 24 h after incision. This treatment also significantly reduced mechanical allodynia (F5,60 = 3.64; P = 0.0061), but the intensity of the analgesia was much less than that of animals that received both a pre-incisional and 24-h dose of Ac-YVAD-CMK (fig. 2B).

To explore the possible site of action of caspase inhibition we determined if local injection of Ac-YVAD-CMK influenced the mechanical allodynia induced by the tissue injury in the hindpaw. The data in figure 2C demonstrates that intraplantar injection of this caspase-1 inhibitor significantly reduced mechanical allodynia by 0.5 h after injection, and completely reversed allodynia by 2 h after injection(F12,105 = 7.37; P < 0.0001).

We next assessed a novel nonpeptide caspase-1 inhibitor, VRTXSD727, in the same model and conditions to confirm that it was in fact caspase-1 inhibition that was leading to the observed effects. Figure 3 shows that oral administration of the compound significantly reduced mechanical sensitivity after incision (F1, 56 = 30.71; P < 0.0001). The efficacy of this orally administered drug was somewhat lower, however.

Fig. 3.

Fig. 3

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Effect of VRTXSD727 on mechanical sensitivity. The compound was administered via oral gavage 2 h before testing. The dosage was 100 mg/kg, twice daily. Values are displayed as mean ± SEM, n = 8, *** p < 0.001 compared to the control group.

Caspase-1 inhibition suppresses thermal hyperalgesia after paw incision

Paw incision induced significant thermal hyperalgesia during the 48 h following incision as displayed in figure 4A. Before treatment the baseline of paw withdrawal latency was 12.04 ± 0.49 and 10.5 ±0.60 s, respectively, in control and Ac-YVAD-CMK group. Paw incision dramatically induced thermal hyperalgesia. However, pretreatment with Ac-YVAD-CMK (10 mg/kg, subcutaneous) 30 min before incision slightly but significantly attenuated this sensitization. The effect reached maximum 2-h postincision, and was sustained for 24 h (fig. 4A). After a second injection of inhibitor at 24 h following incision analgesia was sustained, though not fully reversed as was observed for the effects on mechanical sensitization (F10, 110 = 5.22, P < 0.0001). The injection of Ac-YVAD-CMK one time at 24-h postincision also attenuated thermal hyperalgesia near the incisional site, though the effect was small (fig. 4B).

Fig. 4.

Fig. 4

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on thermal hyperalgesia in the paw incision model. A. Effect of two-injections of Ac-YVAD-CMK on thermal sensitivity. The treatment style was same as described in figure 2. Values are displayed as the mean ± SEM, n = 6, * p < 0.05, ** p < 0.01 or *** p < 0.001 compared to control group. B. Ac-YVAD-CMK injection was made at 24 h post-paw incision.

Caspase-1 inhibition suppresses edema after paw incision

Edema is a common feature of tissue surrounding surgical wounds. We hypothesized that that caspase-1 inhibition would reduce hindpaw edema in the incisional model. Figure 5A–C shows that paw incision significantly induced paw edema in time dependent manner. The paw edema reached peak level 6 h after incision and then declined, but was still present 48 h after incision. Caspase-1 inhibitor pretreatment significantly reduced edema after incision. The effect was detectable 24-h postsurgery (P < 0.001) when the first injection of caspase-1 inhibitor was made 30 min prior to incision. Redosing this compound at 24-h postincision sustained the effect for at least additional 4 h (P < 0.05) as displayed in figure 5A. Administering Ac-YVAD-CMK for the first time 24 h after incision led to a statistically significant reduction in edema as well as demonstrated in figure 5B.

Fig. 5.

Fig. 5

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on paw edema induced by paw surgery. A. Effect of two-injections of Ac-YVAD-CMK on paw edema. The treatment style was same as described in figure 2. Values are displayed as mean ± SEM, n = 6, * p < 0.05 or *** p < 0.001 compared to control group. B. Ac-YVAD-CMK injection was made at 24 h post-paw incision. C. The photo shows paw edema 24 h after paw incision (arrow indication).

Caspase-1 inhibition suppresses paw temperature elevation after paw incision

Local tissue temperature increase after tissue injury is an expected component of the inflammatory response, and contributes to the ongoing activity in nociceptors and ongoing pain in inflammatory models 32–34. Here we tested if incision induced an increase of paw warmth after paw surgery, and the reliance of that increase on caspase-1 activity. The data shows that incision significantly induced local hyperthermia, and this increase was sustained for about 30-h postsurgery (fig. 6). Administration of a caspase-1 inhibitor significantly suppressed the hyperthermia in incised paws.

Fig. 6.

Fig. 6

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on paw temperature after paw incision. Effect of two-injections of Ac-YVAD-CMK on thermal sensitivity. The treatment style was same as described in figure 2. Values are mean ± SEM, n = 6, * p < 0.05 compared to control group.

Caspase-1 inhibition reduces IL-1β, MIP-1α and G-CSF levels in peri-incisional skin tissue

Previous studies demonstrated robust increases in the levels of several cytokines and chemokines in peri-incisional skin tissue 4–7,35. Here we hypothesized that the inhibition of caspase-1 activation would reduce IL-1β levels and perhaps the levels of downstream mediators. The data in figure 7 demonstrate that IL-1β, MIP-1α and G-CSF were increased after incision, and followed time courses similar to those observed in our previous studies. However, caspase-1 inhibition with Ac-YVAD-CMK reduced cytokine levels. Specifically, IL-1β levels were reduced from 2 to 48 h after incision as predicted, though some augmentation of IL-1β levels was still observed. Abundance of the chemokine MIP-1α was significantly reduced 26 to 48 h after incision, and the cytokine G-CSF was significantly reduced from 2 to 26 h as well. Likewise, another caspase-1 inhibitor, VRTXSD727 also reduced IL-1β level after paw incision as shown in figure 8. But the magnitude of IL-1β level was much less than that of Acc-YVAD-CMK.

Fig. 7.

Fig. 7

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on cytokines in incised site. A shows interleukin-1β (IL-1β); B shows macrophage-inflammatory protein 1α (MIP-1α), C shows granulocyte colony stimulating factor (G-CSF). The treatment regimen was same as described in figure 2. Values are mean ± SEM, n = 4 control group; n = 6 Ac-YVAD-CMK group; * p < 0.05, ** p < 0.01 or *** p < 0.001 compared to control group.

Fig. 8.

Fig. 8

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Effect of caspase-1 inhibitor VRTXSD727 on interleukin-1β (IL-1β) in incised site. The treatment regimen was same as described in figure 3. Values are mean ± SEM; n = 8; * p < 0.05 compared to control group.

Caspase-1 inhibition suppresses PGE-2 production in peri-incisional tissue

PGE-2 is a well studied nociceptive mediator. Furthermore, IL-1β stimulates PGE-2 production in other epithelial systems 36. We hypothesized that PGE-2 production would be lowered by caspase-1 inhibition in the incisional model. Because of the relative instability of PGE-2 in tissue, we measured levels of a more stable metabolite (biocyclo PGE-2) to estimate actual PGE-2 production in vivo. Abundance of this metabolite was lower at 26 h after incision if the incised mice were treated with the caspase-1 inhibitor when compared with the control group (fig. 9). With caspase-1 inhibition the level of PGE-2 metabolite was actually below baseline levels 2 h after incision.

Fig. 9.

Fig. 9

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Effect of caspase-1 inhibitor Ac-YVAD-CMK on prostaglandin E2 in incised site. The open bars represent the control group, and the black bars represent the Ac-YVAD-CMK group. Values are mean ± SEM, n = 4 control group; n = 6 Ac-YVAD-CMK group; * p < 0.05 or ** p < 0.01 compared to control group.

Expression of caspase-1 in the wound area

Though we had determined caspase-1 activity to be enhanced in the skin surrounding incisions, and that local inhibition of caspase-1 activity reduced postincisional nociceptive sensitization, we sought to define which cells in the incised skin expressed caspase-1. We found that caspase-1 was expressed in both epidermal keratinocytes (fig. 10A), and in neutrophils infiltrating the dermis and subdermal layers (fig. 10A and B) after incision. Furthermore, the expression of caspase-1 and IL-1β were highly overlapping as demonstrated in triple labeling experiments (fig. 10B). The specificity of IL-1β antibody was confirmed by preabsorption of the antibody with the blocking peptide which abolished staining (data not shown).

Fig. 10.

Fig. 10

Fig. 10

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Immune staining of caspase-1, interleukin-1β (IL-1β) and neutrophils in wound area after incision. Panel A is expression of caspase-1 (green) with IL-1β (red) in keratinocyte of the wound skin. EP: Epidermal layer; Derm: dermal layer. Panel B is triple staining of caspase-1 (green), IL-1β (red) versus neutrophils (blue) in skin dermis layer. Scale bar is 50 µm.

Discussion

Cytokines and other inflammatory mediators are produced in great abundance near incisional wounds and in other pain models characterized by inflammation 4–6,37,38. The cytokine IL-1β is one of the best characterized of these mediators. Intradermal administration of IL-1β into hindpaw skin leads to robust mechanical sensitization 39. Binshtok et al. demonstrated that IL-1β can act directly on nociceptors to support sensitization 40. In this study, the authors found that IL-1β rapidly and directly activated peripheral nociceptors to generate action potentials in afferent neurons and induce nociceptive hypersensitivity. Moreover, using oxazolone to induce inflammation, Wannamaker et al. demonstrated that VX-765, a highly selective caspase-1 inhibitor, could reduce levels of IL-1β in inflamed skin 22 though no nociceptive measurements were made. Thus IL-1β acts as a “sensor” in inflammatory processes; blocking IL-1β production would be expected to be antiinflammatory and analgesic. The enzyme best established to produce active IL-1β from its proform in skin is caspase-1.

We set out in these studies to assess the role of the caspase-1 pathway in postincisional pain and inflammation using behavioral testing coupled with measurements of biochemical mediators. The principal findings of these studies were that (1) caspase-1 inhibition using two systemically administered chemically distinct compounds reduced mechanical allodynia and thermal hyperalgesia induced by hindpaw incision, (2) the local inhibition of caspase-1 after incision reduced sensitization, (3) caspase-1 inhibition reduced other measures of inflammation like temperature increase and edema after incision, (4) caspase-1 inhibition reduced production of IL-1β and several inflammatory mediators of various classes present in incised skin, and (5) caspase-1 activity increased after incision likely from its expression in keratinocytes and infiltrating neurtrophils.

Caspase-1 is one of the best characterized caspases and is best known for its role in inflammation and innate immune responses 41. The activity of caspase-1 is triggered by a cytosolic multiprotein complex, termed the “inflammasome” composed of a neuronal apoptosis inhibitory protein C2TA, HET-E, TP1 domain-, leucine-rich repeat region-, and pyrin domain (NALP) family member, the protein apoptosis associated speck-like protein containing a caspase-recruitment domain and caspase-1 42–45. Two types of inflammasomes which have been identified in skin are NALP1 and NALP2/319,44. They can be activated by danger signals such as bacterial and viral components, Adenosine-5'-triphosphate or uric acid, and act as sensors for the system of innate immunity. Several investigations have demonstrated that caspase-1 and inflammasomes are involved in other inflammatory processes in skin, such as skin contact hypersensitivity18,46, and psoriasis47. Recently both inflammasomes and IL-1β in epidermal keratinocytes were found to support nociceptive sensitization in a rat model of complex regional pain syndrome48. In the current study, we found that caspase-1 production and activity were significantly increased by tissue injury after paw incision. Immunohistochemical study further revealed that caspase-1 was mainly produced in keratinocytes in the epidermal layer and infiltrating neutrophils in dermis and deeper layers in this model. The observation of the co-expression of caspase-1 and IL-1β in these cells suggest they are major sources of cytokine production. However, we should point out that we did not address how caspase-1 activity is regulated in the current work. But the evidence demonstrated that caspase-1 activity to cleave to active IL-1β from its precursor is driven by multiple pathways such as NALP-1, NALP3 or nucleotide-binding oligomerization domain-like receptor C4, absent in melanoma2 49,50. We will pursue these issues in the future study.

The current work provides first evidence that caspase-1 inhibitors can reverse postoperative nociceptive sensitization and inflammation after paw incision. Application of Ac-YVAD-CMK, a specific and permeable caspase-1 inhibitor significantly reduced mechanical allodynia, thermal hyperalgesia and edema in the paw incision model. Our results were similar to the antihyperalgesic effect of an endogenous competitive IL-1 receptor antagonist (Anakinra) observed in a similar mouse paw incisional model 9. Our work goes beyond the existing data, however, by demonstrating a role for IL-1β in supporting the overall inflammation present near incisional wounds, and by demonstrating that local administration of agents blocking IL-1β production and activity reduce sensitization. Prior to this time reports had been provided demonstrating that the inhibition of caspase activity within the central nervous system could reduce nociception in dynorphin-induced hyperalgesia in mice 12. While we cannot rule out the possibility that the inhibition of caspase activity in areas other than the incised paw, including the central nervous system, might have contributed to the analgesic effects observed in these studies, local effects were at least partially responsible (fig. 2C). This conclusion is consistent with our biochemical measurements of increased caspase activity in the skin of the incised hindpaws, and our observations that caspase-1 inhibition reduced IL-1β concentration in the skin near the wounds.

While the inhibition of caspase-1 reduced skin levels of IL-1β after incision, the direct product of caspase-1, levels of several other mediators were also reduced. Complementary studies have demonstrated that caspase-1 inhibition suppresses several cytokines other than IL-1β in rodent models of inflammation 15,22,51,52. The cytokine IL-1β is a strong inducer of other cytokines including tumor necrosis factor alpha, IL-6, and MIP-1α 53–55. It may be reasonable to conclude from our studies that by reducing the production of IL-1β after incision, several downstream mediators produced in skin can also be regulated. Previous data from our laboratory indicate that, again, keratinocytes and neutrophils are the most likely sites of production of cytokines after incision6. In addition to having a direct effect on cellular cytokine production, it is possible that caspase-1 inhibition could reduce neutrophil infiltration into the peri-incisional skin thereby reducing cytokine levels. This mechanism would be consistent with other studies finding reduced inflamed skin myeloperoxidase levels, an index of neutrophil infiltration, after caspase inhibition 22, and studies demonstrating direct and indirect neutrophil chemoattractant properties for IL-1β 56.

PGE-2 is an important mediator relevant to inflammation and pain processing. Clinical studies indicate that surgery induces PGE-2 increases in serum and at the wound site such as after cesarean surgery and hip surgery 4,35. Using an animal thoracic incision surgical model, PGE-2 upregulation in central nervous system tissue and at the wound site was observed 57. To our knowledge PGE-2 production has not been studied previously in the paw incisional model. Our results show that paw incision significantly increased PGE-2, whereas caspase-1 inhibition significantly suppressed this change in PGE-2 levels. Several studies have documented that IL-1β stimulates PGE-2 production through the activationof cyclooxygenase-23,15,58. The cyclooxygenase-2 enzyme is a key enzyme producing PGE-2 from arachidonic acid. It should be noted that that while selective cyclooxygenase-2 and nonselective cyclooxygenase inhibitors clearly reduce some dimensions of postoperative pain, the effects of cyclooxygenase inhibition is modality specific in rodent incisional models. For example, Spofford et al. recently showed that guarding but not simple mechanical or thermal nociceptive sensitization were improved after the administration of the cyclooxygenase inhibitor ketoprofen59. Thus while caspase inhibition did reduce peri-incisional PGE-2 levels, this may not have contributed to the effects of the inhibitor observed for thermal and mechanical sensitization.

Though clearly effective, we did observe that caspase-1 inhibitor did not fully abolish IL-1β production in surgical wounds. Several other investigations have observed caspase-1 inhibitors to only incompletely reduce IL-1β production in in-vitro 60 and in-vivo models 15,61–63. This may indicate that there exists a caspase-1 independent pathway mediating IL-1β production, or simply that the dose of inhibitors used yielded submaximal exposures at the site of action. Pertaining to the former possibility, evidence indicates that pro-IL-1β processing is supported by a panel of noncaspase proteases such as cathepsin G, elastase, matrix metalloproteinases, stratum corneum chymotryptic enzyme, neutrophil- and macrophage-derived serine proteases such as proteinase-364–69. Conceivably, the existence of these alternative pathways implicates that the IL-1β processing is a complex process, may limit the utility of caspase-1 inhibitors as analgesics and antiinflammatory agents. Unclear at this time is whether a strategy to more completely reduce IL-1β signaling by blocking the IL-1β receptor such as that employed by Wolf et al.9 would be more effective than only partially blocking IL-1β production, but at the same time reduce the abundance of several other mediators.

In conclusion, caspase-1 activation was identified as an important mechanism participating in postsurgical nociceptive sensitization and inflammation using an incisional model. Caspase-1 inhibition reversed IL-1β production and the consequent production of several downstream nociceptive mediators as well as other manifestations of acute inflammation like warmth and swelling. Both keratinocytes and neutrophils appear to be involved in caspase-1 and IL-1 β production after incision. In the future, studies might be directed at understanding: 1) how caspase-1 is activated after incision, 2) what the relative contributions of various caspase-1 expressing cells are to IL-1 β levels, and 3) if the reduction of caspase-1 activity and IL-1 β production in humans provides a useful method of reducing pain and inflammation in surgical wounds. Strategies such as caspase inhibition which fundamentally alter the biochemistry of wounds offer unique complementary approaches to standard analgesic techniques used after surgery.

Acknowledgement

We thank Dr. Peter Weber, Ph.D., Associate Director (Vertex Pharmaceuticals (Europe) Limited, Abingdon, United Kingdom) for advice on dosing regimen used for VRTXD727.

This work is attributed to Department of Anesthesiology, Veterans Affairs Palo Alto Health Care System

Support: This work was supported by National Institute of Health: GMO79126 to (JDC) in the Department of Anesthesiology, Veterans Affaire Palo Alto Health Care System, Palo Alto, California, United States.

Footnotes

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Summary Statement: Caspase-1 is a key enzyme that cleaves the pro-interlukin-1β to its active form. The current study demonstrates that inhibition of its activity with specific inhibitors significantly reduced postsurgical sensitization and inflammation through a caspase-1/interlukin-1β dependent mechanism.

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