Bradykinin Receptors Play a Critical Role in the Chronic Post-ischaemia Pain Model

Abstract

Complex regional pain syndrome type-I (CRPS-I) is a chronic painful condition resulting from trauma. Bradykinin (BK) is an important inflammatory mediator required in acute and chronic pain response. The objective of this study was to evaluate the association between BK receptors (B₁ and B₂) and chronic post-ischaemia pain (CPIP) development in mice, a widely accepted CRPS-I model. We assessed mechanical and cold allodynia, and paw oedema in male and female Swiss mice exposed to the CPIP model. Upon induction, the animals were treated with BKR antagonists (HOE-140 and DALBK); BKR agonists (Tyr-BK and DABK); antisense oligonucleotides targeting B₁ and B₂ and captopril by different routes in the model (7, 14 and 21 days post-induction). Here, we demonstrated that treatment with BKR antagonists, by intraperitoneal (i.p.), intraplantar (i.pl.), and intrathecal (i.t.) routes, mitigated CPIP-induced mechanical allodynia and oedematogenic response, but not cold allodynia. On the other hand, i.pl. administration of BKR agonists exacerbated pain response. Moreover, a single treatment with captopril significantly reversed the anti-allodynic effect of BKR antagonists. In turn, the inhibition of BKRs gene expression in the spinal cord inhibited the nociceptive behaviour in the 14th post-induction. The results of the present study suggest the participation of BKRs in the development and maintenance of chronic pain associated with the CPIP model, possibly linking them to CRPS-I pathogenesis.

Keywords: Bradykinin receptor antagonists; Chronic pain; Hyperalgesia; Inflammation; Inflammatory mediators; Kinins.

Fig. 1

Experimental design. a First, we administered selective BKR antagonists targeting B₁ and B₂, DALBK (50 nmol/kg) or HOE-140 (150 nmol/kg), by i.p. route. Mechanical allodynia (von Frey test) was evaluated 7, 10, 14, and 21 days post-induction, while cold allodynia (acetone test) was analysed 8, 11, 15, and 22 days post-ischaemia. b The second step additionally evaluated the effect of selective BKR antagonists DALBK (10 nmol/paw) or HOE-140 (10 nmol/paw) when injected by i.pl. route, as well as the agonists Tyr-BK (10 nmol/paw) and DABK (10 nmol/paw) on CPIP-induced mechanical and cold allodynia. Mice nociception was measured as follows: mechanical allodynia 7, 14 and 21 days post-induction and cold allodynia 8, 15 and 22 days post-ischaemia. c Moreover, we assessed if i.p. injection of ACE inhibitor (captopril, 30 mg/kg), 14 days post-CPIP induction, could exacerbate nociceptive behaviour in CPIP mice and whether DALBK (50 nmol/kg, i.p.) or HOE-140 (150 nmol/kg, i.p.) treatment could inhibit the nociceptive response. In this experiment, only mechanical allodynia was verified on the day 14 post-induction of the disease. d Finally, we investigated if inhibition of BKRs gene expression could modulate nociceptive behaviour in CPIP mice. Mice were treated with ASOs targeting B1R (5′-AGGTTCCTGTGGATGGCGTCCC-3′), and B2R (5′-AGAATTCTGTTCACTGTTTCTTCCCTG-3′), as well as mismatch control (5′-GGTGGAT TTGAGGATTTCGGC -3′) administered by i.t. route for three consecutive days, every 12 h and 12 h before assessment of mechanical allodynia on the day 14 after induction of CPIP. All the animals were exposed to a baseline assessment of mechanical and cold allodynia prior to drug administration

Fig. 2

The anti-oedematogenic effect of the B₂R antagonist HOE-140 (150 nmol/kg, i.p.) on hind paws oedema after CPIP induction. Paw-thickness was measured before and posteriorly to the CPIP induction, specifically during the 8 h following induction. All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 represent the statistical significance between the CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 3

Effect of i.p. administration of the BKR antagonists (HOE-140 or DALBK) on mechanical and cold allodynia during CPIP. The animals were treated with DALBK (150 nmol/kg) or HOE-140 (50 nmol/kg) or vehicle by i.p. route once a day on days 7, 10, 14 and 21, after CPIP induction and evaluated for mechanical allodynia by von Frey test (a–d). Additionally mice were newly treated with DALBK (150 nmol/kg) or HOE-140 (50 nmol/kg) or vehicle, also by i.p. route, once a day on days 8, 11, 15 and 22 after induction and evaluated for cold allodynia by acetone test (e–h). All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.05, and ^###p < 0.001 represent the statistical significance between CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 4

CPIP-induced mechanical and cold allodynia versus i.pl. administration of the BKR antagonists HOE-140 and DALBK. Mice were treated with HOE-140 (10 nmol/paw) or DALBK (10 nmol/paw) or vehicle by i.pl. injection once a day on days 7, 14 and 21 after CPIP induction and evaluated for mechanical allodynia by von Frey test (a–c). On days 8, 15 and 22 after induction, the animals were treated again with DALBK (10 nmol/paw) or HOE-140 (10 nmol/paw) or vehicle, once a day also by i.pl. route, and evaluated for cold allodynia by acetone test (d–f). The red arrows indicate the time of treatment. All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 represent the statistical significance between CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 5

Mice were treated with selective kinin B₁ or B₂ receptor agonists [DABK (20 nmol/paw) or Tyr-BK (10 nmol/paw)] or vehicle, by i.pl. route, once a day on days 7, 14 and 21 post-CPIP induction and evaluated for mechanical allodynia by von Frey test (a–c). Furthermore animals received Tyr-BK (10 nmol/paw) or DABK (20 nmol/paw), also by i.pl. injection, once a day on days 8, 15 and 22 of disease, and were evaluated for cold allodynia by acetone test (d–f). The red arrows indicate the time of treatment. All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 represent the statistical significance between CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 6

Inhibition of BKRs gene expression in vivo positively modulates nociceptive behaviour in CPIP mice. Inhibition of gene expression of the B₁ and B₂ receptors (i.t.) in the mechanical and cold allodynia on the 14th day post-CPIP induction (a and d). Mice were treated with ASOs targeting B₁R (5′-AGGTTCCTGTGGATGGCGTCCC-3′), and B₂R (5′-AGAATTCTGTTCACTGTTTCTTCCCTG-3′), as well as mismatch control (5′-GGTGGAT TTGAGGATTTCGGC -3′) administered by i.t. route for three consecutive days, every 12 h and 12 h before assessment of mechanical and cold allodynia on the day 14 after induction of CPIP (b and e). A single-dose of captopril (30 mg/kg, p.o.) did not exacerbate CPIP-induced mechanical and cold allodynia (c and f). On the 14th-day post-ischaemia, we administered captopril (30 mg/kg, p.o.) with and without selective BKR antagonists (DALBK (50 nmol/kg, i.p.) and HOE-140 (150 nmol/kg, i.p.) and posteriorly evaluated mechanical and cold allodynia by von Frey test (c) and acetone test (f). The red arrows indicate the time of treatment. All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 represent the statistical significance between CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 7

CPIP-induced mechanical and cold allodynia in female mice versus i.p. administration of the BKR antagonists HOE-140 (150 nmol/kg) and DALBK (50 nmol/kg). Mice were treated with BKR antagonists or vehicle once a day on days 7, 10, 14 and 21, after CPIP induction. In these days, mice were evaluated for mechanical allodynia by von Frey test (a–d). Additionally on days 8, 11, 15 and 22 after induction mice were newly treated with BKR antagonists DALBK (50 nmol/kg) or HOE-140 (150 nmol/kg) or vehicle, by i.p. route, once a day and evaluated for cold allodynia by acetone test (e–h). The red arrows indicate the time of treatment. All data are expressed as mean ± SEM of 4–6 animals/group and are representative of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 represent the statistical significance between CPIP mice versus treated mice. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 represent the statistical significance between CPIP group versus sham. Statistical comparison of the data was performed by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 8

BK levels in the tissues 22 days after CPIP induction. Analysis of tissue levels of BK in spinal cord and paw samples from sham animals and untreated CPIP control. ^#p < 0.05 statistical difference between CPIP group versus sham group (one-way ANOVA followed Newmann–Keuls post hoc test)

Fig. 9

Over the years some authors have hypothesised a relationship between BKRs and the pathogenesis of CRPS-I. In our study, using a CPIP animal model, we provide evidence reinforcing the deleterious contribution of B₁ and B₂ receptors to disease development, considering their pro-inflammatory and algesic properties. In this context, BKR activation not only activates the nociceptors but also modulate the release of neuropeptides, including CGRP and substance P, which contributed to neurogenic inflammation, and prostaglandins and NO, which generate the vicious cycle of chronic inflammation and pain. BK is produced in the spinal cord in response to intense peripheral noxious stimuli and acts through BKRs expressed by dorsal horn neurons where PKA, PKC, and ERK-dependent signalling pathways are activated, which mediate NMDA and AMPA-mediated synaptic transmission. Furthermore, the pro-inflammatory mediators released in response to BK also activate microglia and astrocytes, which contribute to the maintenance of the inflammatory state, facilitating pain transmission. Herein, we demonstrated that administration of antisense oligonucleotides (ASOs) targeting BKRs inhibits the pain signalling transmission in spinal cord. The excitatory activity of kinins in pain circuitry in addition to chronic inflammation are linked to maladaptive mechanisms—peripheral and central sensitisation—that altered endogenous pain modulation and consequently pain perception. In turn, we also showed that BKR antagonists administration (HOE-140 and DALBK) showed be able to modulate the nociceptive behaviour. Thus, approaches inhibiting the excitatory actions of BKRs may have clinical relevance for the treatment of neuropathic pain and consequently CRPS-I. PK protein kinase, CGRP calcitonin gene-related peptide, NO nitric oxide, ERK extracellular signal–regulated kinases, NMDA N-methyl-d-aspartate receptor, AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, SP substance P