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Pain Medicine  |   October 2010
Caspase-1 Modulates Incisional Sensitization and Inflammation
Author Affiliations & Notes
  • De-Yong Liang, Ph.D.
    *
  • XiangQi Li, M.D.
  • Wen-Wu Li, Ph.D.
    *
  • Dennis Fiorino, Ph.D.
  • Yanli Qiao, M.D.
    §
  • Peyman Sahbaie, M.D.
    *
  • David C. Yeomans, Ph.D.
  • J. David Clark, M.D., Ph.D.
    #
  • * Research Associate, † Research Assistant, # Professor, Department of Anesthesiology, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, and Department of Anesthesiology, Stanford University School of Medicine, Stanford, California. ‡ Research Fellow, Vertex Pharmaceuticals, Inc., San Diego, California. § Research Assistant, ∥ Associate Professor, Department of Anesthesiology, Stanford University School of Medicine.
Article Information
Pain Medicine / Central and Peripheral Nervous Systems / Pain Medicine
Pain Medicine   |   October 2010
Caspase-1 Modulates Incisional Sensitization and Inflammation
Anesthesiology 10 2010, Vol.113, 945-956. doi:10.1097/ALN.0b013e3181ee2f17
Anesthesiology 10 2010, Vol.113, 945-956. doi:10.1097/ALN.0b013e3181ee2f17
What We Already Know about This Topic
  • ❖ Surgery produces inflammation and pain, and interleukin-1β is a crucial factor for these processes.
  • ❖ Caspase-1 is a key enzyme involved in interleukin-1β production.
What This Article Tells Us That Is New
  • ❖ Caspase-1 inhibitors are effective in this animal model for postoperative pain and may reduce patients' pain and inflammation after surgery.
POSTOPERATIVE pain is an expected consequence of most surgeries. Unfortunately, 30–40% of patients 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, increased levels of many cytokines have been reported in rodents and humans, including interleukin-1β, interleukin-6, tumor necrosis factor-α, granulocyte colony-stimulating factor (G-CSF), macrophage-inflammatory protein-1α (MIP-1α), keratinocyte-derived cytokine, and others.5–7,9,10 The archetypical proinflammatory cytokine interleukin-1β has been implicated strongly in supporting pain during inflammation and after tissue injury.5,6,9,11–13 Furthermore, administration of an interleukin-1 receptor antagonist sharply reduces nociceptive sensitization surrounding incisions in rodents.9 Beyond its direct sensitizing effects, interleukin-1β induces the production of other proinflammatory cytokines and mediators linked to nociception, including tumor necrosis factor-α, interleukin-6, cyclooxygenase-2, chemokines, substance P, and nerve growth factor.14–17 Very little is known, however, about the mechanisms supporting interleukin-1β production and release after incision.
Caspase-1 (also known as interleukin-1β-converting enzyme) is a cysteine protease that cleaves prointerleukin-1β, prointerleukin-18, and prointerleukin-33 to form 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 1 s 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 interleukin-1β production, nociceptive sensitization, edema, warmth, and the generation of inflammatory mediators downstream from interleukin-1β, which also supports 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 four 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 and then diluted in 0.9% NaCl before use. For local intraplantar injection, Ac-YVAD-CMK was injected in a volume of 10 μl of 0.9% NaCl/2.5% dimethyl sulfoxide final concentration at 24 h after incision. Systemic administration involved the subcutaneous injection of 100 μl NaCl of the same solution. In some experiments, animals received the first treatment 30 min before incision and the second treatment 24 h after incision. 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 10 ml/kg, twice daily. Oral administration of VRTXSD727 was performed 2 h before behavioral testing each day.
Surgical Preparation
Paw incision in mice was performed in our laboratory as described in previous studies.5–7 Briefly, the mice were anesthetized with isoflurane delivered via  a nose cone. After sterile preparation of the right hind paw, 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 increased 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 incisions 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 have 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 g were applied to the plantar surface of the right hind paw 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 hind paw 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 toward the same area of the hind paw 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.
Hind Paw Edema
Paw thickness from dorsal side to ventral side was measured by a laser sensor technique as described previously by Guo and et al  .,28 as modified for mice by Clark and et al  .5 Briefly, the mice were first anesthetized by exposure to isoflurane. Each hind paw 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 hind paws. The ratio of these thickness measurements was used to compare mice.
Hind Paw Temperature
The temperature of the hind paw was measured using a fine wire thermocouple (Omega, Stamford, CT) applied to the paw skin as previously described in rats by Guo et al  .28 Briefly, the investigator held the device using an insulating Styrofoam block. Three sites were tested twice each over the dorsum of the hind paw: 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 hind paw, the same site was immediately tested on the contralateral hind paw. The six measurements for each hind paw 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 the incision, with approximate 1.5-mm 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 −80°C until analysis. For use, these samples were first cut into small pieces with microscissors and 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 E2Assays
Cytokines interleukin-1β, MIP-1α, and G-CSF were analyzed by BioPlex mouse cytokine assay (Bio-Rad Laboratories) as described previously.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 the 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 being washed 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.
The prostaglandin E2level was estimated by enzyme-linked immunosorbent assay measurement of the prostaglandin E2metabolite. This assay was conducted according to 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 prostaglandin E2and its intermediate metabolites to a stable prostaglandin E2metabolite. The concentration of prostaglandin E2metabolite in the samples was determined using a specific enzyme-linked immunosorbent assay. The metabolite assay was chosen because prostaglandin E2is 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 manufacturer's protocol obtained from BioVision (Mountain View, CA). Briefly, a volume of 50 μl of protein sample from skin tissue was added to 50 μl of 2× caspase-1 reaction buffer (containing 10 mm dithiothreitol) in a duplicate manner in a 96-well plate. Then, 1 mm 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 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, the mice were sacrificed using carbon dioxide asphyxiation, which was followed by intracardiac perfusion of 20 ml of 0.9% NaCl followed by 20 ml of 10% neutrally buffered formalin. The hind paws were then removed and incubated in 10% buffered formalin for 24 h. After overnight decalcification, the tissue was processed for paraffin sectioning in an automated fashion (Tissue Tek VIP; Miles Scientific, Naperville, IL). After 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, interleukin-1β, or neurophils overnight at 4°C in milk–Tris-buffered saline. The primary antibodies included polyclonal antiinterleukin-1β, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anticleaved (mature) caspase-1, 1:200 (Abcam Inc., Cambridge, MA), and antimouse neutrophil antibody, 1:5000 (ABD Serotec, Kidlington, Oxford, United Kingdom). For specificity of interleukin-1β antibody, the preabsorption of the antibody with blocking peptide was conducted before adding to the section. Sections were then rinsed and transferred to milk–Tris-buffered saline containing fluorescein-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 microscopy 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 led 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 ANOVA followed by Bonferroni post hoc  test for multiple comparisons. For simple comparisons of two groups, a two-tailed Student t  test 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 interleukin-1β production. We first directly assessed this enzyme activity in the skin surrounding incisions in mice. Figure 1shows that caspase-1 activity was significantly and transiently increased after paw incision.
Fig. 1.  Incision enhances caspase-1 activity after incision. The data are expressed as the fold change relative to control. Values expressed as mean ± SEM, n = 6, ***P  < 0.001 compared with nonincision group (t = 0 h).
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Fig. 1.  Incision enhances caspase-1 activity after incision. The data are expressed as the fold change relative to control. Values expressed as mean ± SEM, n = 6, ***P  < 0.001 compared with nonincision group (t = 0 h).
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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 in figure 2A. Systemic administration of Ac-YVAD-CMK 30 min before incision reduced the allodynia, with maximal effectiveness 1 to 2 h after incision and 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.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on mechanical sensitivity in the 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 after paw incision. (C  ) Effect of Ac-YVAD-CMK local intraplantar injection on mechanical sensitivity 24 h after paw incision. Solid line  is Ac-YVAD-CMK group, dashed line  is control group. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 nonincision group compared with the control group; ## P  < 0.01 or ### P  < 0.001 comparing Ac-YVAD-CMK group with the control group.
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Fig. 2.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on mechanical sensitivity in the 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 after paw incision. (C  ) Effect of Ac-YVAD-CMK local intraplantar injection on mechanical sensitivity 24 h after paw incision. Solid line  is Ac-YVAD-CMK group, dashed line  is control group. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 nonincision group compared with the control group; ## P  < 0.01 or ### P  < 0.001 comparing Ac-YVAD-CMK group with the control group.
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To explore the unexpected efficacy of repeated inhibitor administration further, we conducted a separate experiment in which Ac-YVAD-CMK was first administered to incised animals 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 preincisional and 24-h dose of Ac-YVAD-CMK (fig. 2B).
To explore the possible site of action of caspase inhibition, we determined whether local injection of Ac-YVAD-CMK influenced the mechanical allodynia induced by the tissue injury in the hind paw. The data in figure 2Cdemonstrate 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 3shows 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.  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 with the control group.
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Fig. 3.  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 with the control group.
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Caspase-1 Inhibition Suppresses Thermal Hyperalgesia after Paw Incision
Paw incision induced significant thermal hyperalgesia during the 48 h after 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 the control and the 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 at 2 h after incision and was sustained for 24 h (fig. 4A). After a second injection of inhibitor at 24 h after incision, analgesia was sustained, although 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 after incision also attenuated thermal hyperalgesia near the incisional site, although the effect was small (fig. 4B).
Fig. 4.  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 the 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 with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision.
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Fig. 4.  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 the 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 with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision.
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Caspase-1 Inhibition Suppresses Edema after Paw Incision
Edema is a common feature of tissue surrounding surgical wounds. We hypothesized that caspase-1 inhibition would reduce hind paw edema in the incisional model. Figure 5A–Cshows that paw incision significantly induced paw edema in a time-dependent manner. The paw edema reached its 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 after surgery (P  < 0.001) when the first injection of caspase-1 inhibitor was made 30 min before incision. Redosing this compound at 24 h after incision sustained the effect for at least an 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 is demonstrated in figure 5B.
Fig. 5.  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 the same as described in figure 2. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 compared with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision. (C  ) The photo shows paw edema 24 h after paw incision (arrow  indication).
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Fig. 5.  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 the same as described in figure 2. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 compared with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision. (C  ) The photo shows paw edema 24 h after paw incision (arrow  indication).
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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 it contributes to the ongoing activity in nociceptors and ongoing pain in inflammatory models.32–34 Here, we tested whether incision induced an increase in paw warmth after paw surgery, and the reliance of that increase on caspase-1 activity. The data show that incision significantly induced local hyperthermia, and this increase was sustained for about 30 h after surgery (fig. 6). Administration of a caspase-1 inhibitor significantly suppressed the hyperthermia in the incised paws.
Fig. 6.  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 the same as described in figure 2. Values are mean ± SEM, n = 6, *P  < 0.05 compared with control group.
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Fig. 6.  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 the same as described in figure 2. Values are mean ± SEM, n = 6, *P  < 0.05 compared with control group.
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Caspase-1 Inhibition Reduces Interleukin-1β, MIP-1α, and G-CSF Levels in Periincisional Skin Tissue
Previous studies demonstrated robust increases in the levels of several cytokines and chemokines in periincisional skin tissue.4–7,35 Here, we hypothesized that the inhibition of caspase-1 activation would reduce interleukin-1β levels and perhaps the levels of downstream mediators. The data in figure 7demonstrate that interleukin-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, interleukin-1β levels were reduced from 2 to 48 h after incision as predicted, although some augmentation of interleukin-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 interleukin-1β level after paw incision as shown in figure 8. However, the magnitude of interleukin-1β reduction was much less than that with Ac-YVAD-CMK.
Fig. 7.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on cytokines in incised site. (A  ) Interleukin-1β (IL-1β). (B  ) Macrophage-inflammatory protein-1α (MIP-1α). (C  ) Granulocyte colony-stimulating factor (G-CSF). The treatment regimen was the 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 with control group.
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Fig. 7.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on cytokines in incised site. (A  ) Interleukin-1β (IL-1β). (B  ) Macrophage-inflammatory protein-1α (MIP-1α). (C  ) Granulocyte colony-stimulating factor (G-CSF). The treatment regimen was the 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 with control group.
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Fig. 8.  Effect of caspase-1 inhibitor VRTXSD727 on interleukin-1β (IL-1β) in incised site. The treatment regimen was the same as described in figure 3. Values are mean ± SEM; n = 8; *P  < 0.05 compared with control group.
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Fig. 8.  Effect of caspase-1 inhibitor VRTXSD727 on interleukin-1β (IL-1β) in incised site. The treatment regimen was the same as described in figure 3. Values are mean ± SEM; n = 8; *P  < 0.05 compared with control group.
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Caspase-1 Inhibition Suppresses Prostaglandin E2Production in Periincisional Tissue
Prostaglandin E2is a well studied nociceptive mediator. Furthermore, interleukin-1β stimulates prostaglandin E2production in other epithelial systems.36 We hypothesized that prostaglandin E2production would be lowered by caspase-1 inhibition in the incisional model. Because of the relative instability of prostaglandin E2in tissue, we measured levels of a more stable metabolite (biocyclo prostaglandin E2) to estimate actual prostaglandin E2production 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 prostaglandin E2metabolite was actually below baseline levels 2 h after incision.
Fig. 9.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on prostaglandin E2in incised site. The open bars  represent the control group, and the filled 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 with control group.
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Fig. 9.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on prostaglandin E2in incised site. The open bars  represent the control group, and the filled 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 with control group.
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Expression of Caspase-1 in the Wound Area
Although we had determined caspase-1 activity to be enhanced in the skin surrounding the 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 interleukin-1β was highly overlapping, as demonstrated in triple labeling experiments (fig. 10B). The specificity of interleukin-1β antibody was confirmed by preabsorption of the antibody with the blocking peptide which abolished staining (data not shown).
Fig. 10.  Immune staining of caspase-1, interleukin-1β (IL-1β), and neutrophils in wound area after incision. (A  ) Expression of caspase-1 (green  ) with interleukin-1β (red  ) in keratinocyte of the wound skin. EP = epidermal layer; Derm = dermal layer. (B  ) Triple staining of caspase-1 (green  ), interleukin-1β (red  ) versus  neutrophils (blue  ) in skin dermis layer. Scale bar  is 50 μm.
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Fig. 10.  Immune staining of caspase-1, interleukin-1β (IL-1β), and neutrophils in wound area after incision. (A  ) Expression of caspase-1 (green  ) with interleukin-1β (red  ) in keratinocyte of the wound skin. EP = epidermal layer; Derm = dermal layer. (B  ) Triple staining of caspase-1 (green  ), interleukin-1β (red  ) versus  neutrophils (blue  ) in skin dermis layer. Scale bar  is 50 μm.
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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 interleukin-1β is one of the best characterized of these mediators. Intradermal administration of interleukin-1β into hind paw skin leads to robust mechanical sensitization.39 Binshtok et al  . demonstrated that interleukin-1β can act directly on nociceptors to support sensitization.40 In this study, the authors found that interleukin-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 interleukin-1β in inflamed skin,22 although no nociceptive measurements were made. Thus interleukin-1β acts as a “sensor” in inflammatory processes; blocking interleukin-1β production would be expected to be antiinflammatory and analgesic. The enzyme best established to produce active interleukin-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 hind paw incision, (2) the local inhibition of caspase-1 after incision reduced sensitization, (3) caspase-1 inhibition reduced other measures of inflammation such as temperature increase and edema after incision, (4) caspase-1 inhibition reduced production of interleukin-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 neutrophils.
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 NALP2/3.19,44 They can be activated by danger signals such as bacterial and viral components, or 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 psoriasis.47 Recently, both inflammasomes and interleukin-1β in epidermal keratinocytes were found to support nociceptive sensitization in a rat model of complex regional pain syndrome.48 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 coexpression of caspase-1 and interleukin-1β in these cells suggests they are major sources of cytokine production. We should point out that we did not address in the current work how caspase-1 activity is regulated. However, the evidence demonstrated that caspase-1 activity to cleave to active interleukin-1β from its precursor is driven by multiple pathways such as NALP-1, NALP-3, or nucleotide-binding oligomerization domain-like receptor C4, absent in melanoma2.49,50 We will pursue these issues in a 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 interleukin-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 interleukin-1β in supporting the overall inflammation present near incisional wounds and by demonstrating that local administration of agents blocking interleukin-1β production and activity reduce sensitization. Prior reports have demonstrated that the inhibition of caspase activity within the central nervous system could reduce nociception in dynorphin-induced hyperalgesia in mice.12 Although 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 hind paws, and our observations that caspase-1 inhibition reduced interleukin-1β concentration in the skin near the wounds.
Although the inhibition of caspase-1 reduced skin levels of interleukin-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 interleukin-1β in rodent models of inflammation.15,22,51,52 The cytokine interleukin-1β is a strong inducer of other cytokines, including tumor necrosis factor-α, interleukin-6, and MIP-1α.53–55 It may be reasonable to conclude from our studies that by reducing the production of interleukin-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 incision.6 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 interleukin-1β.56 
Prostaglandin E2is an important mediator relevant to inflammation and pain processing. Clinical studies indicate that surgery induces prostaglandin E2increases in serum and at the wound site, such as after cesarean surgery and hip surgery.4,35 Using an animal thoracic incision surgical model, prostaglandin E2up-regulation in central nervous system tissue and at the wound site has been observed.57 To our knowledge, prostaglandin E2production has not been studied previously in the paw incisional model. Our results show that paw incision significantly increased prostaglandin E2, whereas caspase-1 inhibition significantly suppressed this change in prostaglandin E2levels. Several studies have documented that interleukin-1β stimulates prostaglandin E2production through the activation of cyclooxygenase-2.3,15,58 The cyclooxygenase-2 enzyme is a key enzyme producing prostaglandin E2from arachidonic acid. It should be noted that that although selective cyclooxygenase-2 and nonselective cyclooxygenase inhibitors clearly reduce some dimensions of postoperative pain, the effects of cyclooxygenase inhibition are 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 ketoprofen.59 Thus, although caspase inhibition did reduce periincisional prostaglandin E2levels, this may not have contributed to the effects of the inhibitor observed for thermal and mechanical sensitization.
Although clearly effective, we did observe that caspase-1 inhibitors did not fully abolish interleukin-1β production in surgical wounds. Several other investigations have observed caspase-1 inhibitors to reduce only incompletely interleukin-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 interleukin-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 prointerleukin-1β processing is supported by a panel of noncaspase proteases such as cathepsin G, elastase, matrix metalloproteinases, stratum corneum chymotryptic enzyme, and neutrophil- and macrophage-derived serine proteases such as proteinase-3.64–69 Conceivably, the existence of these alternative pathways implicates that the interleukin-1β processing is a complex process, which may limit the utility of caspase-1 inhibitors as analgesics and antiinflammatory agents. It is unclear at this time whether a strategy to reduce interleukin-1β signaling more completely by blocking the interleukin-1β receptor, such as that employed by Wolf et al  .,9 would be more effective than only partially blocking interleukin-1β production, but at the same time reducing 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 interleukin-1β production and the consequent production of several downstream nociceptive mediators, as well as other manifestations of acute inflammation such as warmth and swelling. Both keratinocytes and neutrophils appear to be involved in caspase-1 and interleukin-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 interleukin-1 β levels, and (3) whether the reduction of caspase-1 activity and interleukin-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.
The authors thank Peter Weber, Ph.D., Associate Director, Vertex Pharmaceuticals (Europe) Limited, Abingdon, United Kingdom, for advice on the dosing regimen used for VRTXD727.
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Fig. 1.  Incision enhances caspase-1 activity after incision. The data are expressed as the fold change relative to control. Values expressed as mean ± SEM, n = 6, ***P  < 0.001 compared with nonincision group (t = 0 h).
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Fig. 1.  Incision enhances caspase-1 activity after incision. The data are expressed as the fold change relative to control. Values expressed as mean ± SEM, n = 6, ***P  < 0.001 compared with nonincision group (t = 0 h).
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Fig. 2.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on mechanical sensitivity in the 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 after paw incision. (C  ) Effect of Ac-YVAD-CMK local intraplantar injection on mechanical sensitivity 24 h after paw incision. Solid line  is Ac-YVAD-CMK group, dashed line  is control group. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 nonincision group compared with the control group; ## P  < 0.01 or ### P  < 0.001 comparing Ac-YVAD-CMK group with the control group.
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Fig. 2.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on mechanical sensitivity in the 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 after paw incision. (C  ) Effect of Ac-YVAD-CMK local intraplantar injection on mechanical sensitivity 24 h after paw incision. Solid line  is Ac-YVAD-CMK group, dashed line  is control group. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 nonincision group compared with the control group; ## P  < 0.01 or ### P  < 0.001 comparing Ac-YVAD-CMK group with the control group.
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Fig. 3.  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 with the control group.
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Fig. 3.  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 with the control group.
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Fig. 4.  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 the 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 with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision.
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Fig. 4.  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 the 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 with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision.
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Fig. 5.  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 the same as described in figure 2. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 compared with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision. (C  ) The photo shows paw edema 24 h after paw incision (arrow  indication).
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Fig. 5.  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 the same as described in figure 2. Values are displayed as mean ± SEM, n = 6, *P  < 0.05 or ***P  < 0.001 compared with control group. (B  ) Ac-YVAD-CMK injection was made at 24 h after paw incision. (C  ) The photo shows paw edema 24 h after paw incision (arrow  indication).
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Fig. 6.  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 the same as described in figure 2. Values are mean ± SEM, n = 6, *P  < 0.05 compared with control group.
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Fig. 6.  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 the same as described in figure 2. Values are mean ± SEM, n = 6, *P  < 0.05 compared with control group.
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Fig. 7.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on cytokines in incised site. (A  ) Interleukin-1β (IL-1β). (B  ) Macrophage-inflammatory protein-1α (MIP-1α). (C  ) Granulocyte colony-stimulating factor (G-CSF). The treatment regimen was the 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 with control group.
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Fig. 7.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on cytokines in incised site. (A  ) Interleukin-1β (IL-1β). (B  ) Macrophage-inflammatory protein-1α (MIP-1α). (C  ) Granulocyte colony-stimulating factor (G-CSF). The treatment regimen was the 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 with control group.
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Fig. 8.  Effect of caspase-1 inhibitor VRTXSD727 on interleukin-1β (IL-1β) in incised site. The treatment regimen was the same as described in figure 3. Values are mean ± SEM; n = 8; *P  < 0.05 compared with control group.
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Fig. 8.  Effect of caspase-1 inhibitor VRTXSD727 on interleukin-1β (IL-1β) in incised site. The treatment regimen was the same as described in figure 3. Values are mean ± SEM; n = 8; *P  < 0.05 compared with control group.
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Fig. 9.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on prostaglandin E2in incised site. The open bars  represent the control group, and the filled 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 with control group.
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Fig. 9.  Effect of caspase-1 inhibitor Ac-YVAD-CMK on prostaglandin E2in incised site. The open bars  represent the control group, and the filled 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 with control group.
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Fig. 10.  Immune staining of caspase-1, interleukin-1β (IL-1β), and neutrophils in wound area after incision. (A  ) Expression of caspase-1 (green  ) with interleukin-1β (red  ) in keratinocyte of the wound skin. EP = epidermal layer; Derm = dermal layer. (B  ) Triple staining of caspase-1 (green  ), interleukin-1β (red  ) versus  neutrophils (blue  ) in skin dermis layer. Scale bar  is 50 μm.
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Fig. 10.  Immune staining of caspase-1, interleukin-1β (IL-1β), and neutrophils in wound area after incision. (A  ) Expression of caspase-1 (green  ) with interleukin-1β (red  ) in keratinocyte of the wound skin. EP = epidermal layer; Derm = dermal layer. (B  ) Triple staining of caspase-1 (green  ), interleukin-1β (red  ) versus  neutrophils (blue  ) in skin dermis layer. Scale bar  is 50 μm.
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