Role of peripheral endothelin receptors in an animal model of complex regional pain syndrome type 1 (CRPS-I)

Abstract

Chronic post-ischemic pain (CPIP) is an animal model of CRPS-I developed using a 3-h ischemia-reperfusion injury of the rodent hind paw. The contribution of local endothelin to nociception has been evaluated in CPIP mice by measuring sustained nociceptive behaviors (SNBs) following intraplantar injection of endothelin-1 or -2 (ET-1, ET-2). The effects of local BQ-123 (ETA-R antagonist), BQ-788 (ETB-R antagonist), IRL-1620 (ETB-R agonist) and naloxone (opioid antagonist) were assessed on ET-induced SNBs and/or mechanical and cold allodynia in CPIP mice. ETA-R and ETB-R expression was assessed using immunohistochemistry and Western blot analysis. Compared to shams, CPIP mice exhibited hypersensitivity to local ET-1 and ET-2. BQ-123 reduced ET-1- and ET-2-induced SNBs in both sham and CPIP animals, but not mechanical or cold allodynia. BQ-788 enhanced ET-1- and ET-2-induced SNBs in both sham and CPIP mice, and cold allodynia in CPIP mice. IRL-1620 displayed a non-opioid anti-nociceptive effect on ET-1- and ET-2-induced SNBs and mechanical allodynia in CPIP mice. The distribution of ETA-R and ETB-R was similar in plantar skin of sham and CPIP mice, but both receptors were over-expressed in plantar muscles of CPIP mice. This study shows that ETA-R and ETB-R have differing roles in nociception for sham and CPIP mice. CPIP mice exhibit more local endothelin-induced SNBs, develop a novel local ETB-R agonist-induced (non-opioid) analgesia, and exhibit over-expression of both receptors in plantar muscles, but not skin. The effectiveness of local ETB-R agonists as anti-allodynic treatments in CPIP mice holds promise for novel therapies in CRPS-I patients.

Fig. 1. Dose response curve of ET-induced SNB

Two days after I/R injury, sham and CPIP mice received a 10 μl i.pl. injection of (A) endothelin-1 (ET-1, from 0.3 pmol to 400 pmol or vehicle), (B) endothelin-2 (ET-2, from 2 pmol to 600 pmol) or (C) vehicle. The x-axis is expressed as the log dose in pmol. The total sustained nociceptive behavior score (tot SNB score) was determined over a 30 minute time period. The EC₅₀ values were calculated using a non-linear regression profile for the curve fit (Prim 4.0), and data are expressed EC₅₀ in pmol (+/− 95% Confidence Intervals). N=7–9/group. Dose response curves of the SNBs to both ET-1 and ET-2 were shifted to the left in CPIP, as compared to sham, mice.

Fig. 2. Effect of ETB-R agonist or ETA/B-R antagonists on ET-induced SNB

Two days after I/R injury, sham (A and B) and CPIP (C and D) mice received a 10 μl i.pl. injection of vehicle (white histogram), BQ-123 (light grey histogram, 5 or 10 nmol, ETA-R antagonist), BQ-788 (dark grey histogram, 30 or 60 nmol, ETB-R antagonist) or IRL-1620 (black histogram, 50 or 200 pmol, ETB-R agonist). Twenty minutes later, they received a 10 μl i.pl. injection of endothelin-1 (ET-1, A: 70 pmol for sham and C: 7 pmol for CPIP) or endothelin-2 (ET-2, B: 200 pmol for sham and D: 125 pmol for CPIP). The total sustained nociceptive behavior score (SNB score) was determined over a period of 30 minutes. N=7–8/group (* p<0.05, ** p<0.01, one-way ANOVA followed by a Dunnett’s t-test). BQ-123 and IRL-1620 reduced, and BQ-788 enhanced, ET-1/2-induced SNBs in CPIP mice, while BQ-123 also reduced ET1/2-induced SNBs in shams.

Fig. 3. Effect of ETB-R agonist or ETA/B-R antagonists on mechanical and cold allodynia

Two days after I/R injury, mechanical (A, B and C) and cold (D, E, F) sensitivities were assessed in both sham and CPIP mice: before, 30 and 60 min after mice received a local 10 μl i.pl. treatment with vehicle, BQ-123 (10 nmol), BQ-788 (60 nmol) or IRL-1620 (50 pmol). N=7–8 / group. Only IRL-1620 significantly elevated the 50% threshold in CPIP mice (* p<0.05, ** p<0.001, One-way ANOVA followed by a Dunnett for multiple comparison test).

Fig. 4. Effect of naloxone on the anti-allodynic effect of IRL-1620 in CPIP mice

Top graph: two days after I/R injury, baseline mechanical sensitivity was assessed in four groups of CPIP mice (patterned histogram). Then, mice received a 5 μl i.pl. injection of naloxone (100 nmol) or vehicle, followed 5 min later by a 5 μl i.pl. injection of IRL-1620 (50 pmol) or vehicle. Mechanical sensitivity was assessed 30 min after the second treatment (black histogram). Bottom graph: the percentage of analgesia was calculated as the relative change between the two measures for each group. The significant elevation of the 50% threshold (A), or % analgesia (B), by IRL-1620 in CPIP mice was unaffected by naloxone treatment. N=7–8 / group (* p<0.05, paired t-test (compared to the value before treatment; ## p<0.05, ### p<0.01, 2-way ANOVA followed by a Bonferroni’s multiple comparison test).

Fig. 5. Distribution of ETA-R and ETB-R in the skin of sham and CPIP mice

Two days post I/R injury, the skin of sham (left panels) and CPIP (right panels) mice was incubated with anti-ETA-R (1:4000, top panels, green) or anti-ETB-R (1:2000, bottom panels, green) antibodies, or stained with hemathoxiline and eosin (H&E). All pictures were taken with a 40 X objective. ETA-R staining was predominantly found in the deeper stratum basalis (s.b.) layer and ETB-R in the medium strati granulosum (s.g.) and spinosum (s.s.) layers. No or poor staining was observed in the external stratum corneum (s.c.) There are no obvious changes in ETA-R or ETB-R staining between sham and CPIP mice.

Fig. 6. Fine cellular distribution of ETA-R and ETB-R in the epidermis and peripheral nerves of sham and CPIP mice

Two days post I/R injury, the epidermis (A) and deep nerves (B) of the skin of sham (left column) and CPIP (right column) mice was incubated with anti-NF200 antibody (1:6000) and anti-ETA-R (1:4000) or anti-ETB-R (1:2000) antibodies. All pictures were taken with a 60 X objective. ETA-R staining was observed at the surface membrane of the keratinocytes from the s.b. layer. ETB-R staining appears within s.g. and s.s. layers, with more intensively stained star-shaped cells found mainly in the s.s. layer, which are most likely Langerhans cells (arrow in A). ETA-R and ETB-R staining was also observed in filamentous structures co-migrating within the NF200-IR fiber bundle. Finally, ETA-R staining was observed on blood vessels (arrows) within the bundle. In the epidermis and in deeper peripheral nerves, ETA-R and ETB-R immunoreactivity shows a similar pattern of distribution in CPIP and sham mice.

Fig. 7. Quantification of ETA-R and ETB-R in the skin and muscle of sham and CPIP mice

Western blot analysis of plantar skin and muscles extracts from sham and CPIP mice. The top membrane was incubated with an anti-ETA-R antibody (1:200, top picture; β-actin, bottom picture), and the bottom membrane was incubated with an anti-ETB-R antibody (1:200, top picture; β-actin, bottom picture). The five first lanes received supernatant from homogenized muscles; the four last lanes received supernatant from homogenized skin. A) The same lane reflects extracts from the same animal. B and C show the quantification by densitometry of the pixel intensity relative to β-actin for ETA-R and ETB-R for both plantar skin (B) and muscle (C). There was a significant upregulation of both ETA-R and ETB-R in CPIP muscle (A & C), but not skin (A & B) (* p < 0.05, ** P < 0.01 unpaired t-test).