Figure 1.
Nociception and oedema induced by i.pl. injection of PNV in mice.
(A–B) Time-course (A) and dose-response (B) curves for the ongoing nociception, measured as an increase in amount of time spent licking the injected paw, induced by PNV. (C–D) Time-course (C) and sum (Σ, D) of the pain-related behaviour scores from 0 to 240 minutes after PNV injection. (E) Time-course of the paw oedema caused by i.pl. injection of PNV in mice. Each point or bar on the curve represents the mean±S.E.M or the medians and interquartile ranges of 5–7 animals. The asterisks denote the significance levels. **P<0.01, ***P<0.001 compared with the PBS group (A, E: two-way ANOVA followed Bonferroni's test, B: one-way ANOVA followed by Dunnett's test, C, D: Mann-Whitney U test).
Figure 2.
Clinically used analgesics reduced PNV nociception.
The effect of systemic pre- (A–D) or post-treatment (G–J) with dipyrone (500 mg/kg, i.p., A and G), morphine (10 mg/kg, i.p., B and H), acetaminophen (400 mg/kg, i.p., C and I) or indomethacin (30 mg/kg, i.p., D and J) on PNV-induced (3 µg/paw) nociception in mice. The effect of local co-administration (E–F) or post-treatment (K–M) with lidocaine (0.4 µmol/paw, i.pl., E and K) or arachnid antivenin (ArAv 1∶30, i.pl., F and M) on PNV-induced (3 µg/paw) nociception. Each column represents the mean or median of 6–8 animals, and the vertical lines show the S.E.M. or interquartile ranges *P<0.05, **P<0.01 ***P<0.001 compared with the vehicle group. A–E: Student's t-test and G–M: Mann-Whitney U test, F: one-way ANOVA followed by Dunnett's test.
Figure 3.
Effect of boiling or dialysing venom in the PNV-induced nociception.
The nociceptive effect produced by i.pl. injection of boiled (A) or dialysed (B) PNV (3 µg/paw) in mice. Each column represents the mean± S.E.M of 6 mice. The asterisks denote the significance levels. **P<0.01, ***P<0.001 compared with the PBS group. #P<0.05, ##P<0.01 compared with the non-boiled or non-dialysed PNV group (one-way ANOVA followed by the Student-Newman-Keuls test).
Table 1.
Amount of histamine, serotonin and glutamate in PNV.
Figure 4.
Role of glutamate, histamine, serotonin and mast cells in the nociceptive effect triggered by PNV.
The effect of i.pl. treatment with the AMPA/kainate receptor antagonist DNQX (1 nmol/paw, A), the NMDA receptor antagonist MK-801 (1 nmol/paw, B), the 5-HT receptor antagonist metysergide (10 nmol/paw, C), the 5-HT4 receptor antagonist GR113808 (15 nmol/paw, D), the 5-HT3 receptor antagonist ondansetron (30 nmol/paw, E), the H1 receptor antagonist promethazine (3 nmol/paw, i.pl., F) or pre-treatment with compound 48/80 (1, 3, 10 and 10 µg/paw, i.pl., G) on PNV-induced (3 µg/paw, i.pl.) nociception in mice. Each column represents the mean± S.E.M of 6–8 mice. The asterisks denote the significance levels. *P<0.05 compared with the vehicle group (Student's t-test).
Figure 5.
The tissue kallikrein-like activity of PNV in vitro.
The time-course (A) and concentration-response (B) curves of tissue kallikrein-like activity in PNV. (C) The substrate (D-Val-Leu-Arg p-nitroaniline, 0.09–3.00 mM) concentration curve for the kallikrein-like activity of PNV (150 µg/ml). (D) Kinin detection after the incubation of PNV (150 mg/ml) with human high- (HMWK) or low- (LMWK) molecular-weight kininogen (200 nM). (E) The kallikrein-like activity of PNV in the presence or absence of the plasma kallikrein inhibitor SBTI (3 µg/ml) or the tissue kallikrein inhibitor aprotinin (10 µg/ml) or after boiling. Each point or bar represents the mean±S.E.M of 3 experiments carried out in duplicate. The asterisks denote the significance levels. *P<0.05, ***P<0.001, compared with the vehicle group. ###P<0.001 compared with the vehicle plus PNV group (one-way ANOVA followed by the Student-Newman-Keuls test).
Figure 6.
Role of tissue kallikrein and kinins in PNV-induced nociception.
The effect of i.pl. treatment with the kallikrein inhibitors aprotinin (10 µg/paw, A) and SBTI (3 µg/paw, B) or the B2 receptor antagonist HOE140 (3 nmol/paw, D) on PNV-induced (3 µg/paw, i.pl.) nociception in mice. Each column represents the mean± S.E.M of 5–6 mice (A, B and C). The asterisks denote the significance levels. *P<0.05, **P<0.01 compared with the PNV group (A, B, C: Student's t-test).
Figure 7.
Involvement of TRPV1, ASIC, cyclooxygenase or sodium channels in PNV-induced nociception.
The effect of i.pl. treatment with the acid-sensitive ion channel (ASIC) blocker amiloride (100 nmol/paw, A), the cyclooxygenase inhibitor indomethacin (30 µmol/paw, B), the sodium channel blocker tetrodotoxin (TTX) (20 pmol/paw, C) or the selective TRPV1 antagonist SB366791 (1 nmol/paw, D) on PNV-induced (3 µg/paw, i.pl.) nociception in mice. E) The specific binding of [3H]-resiniferatoxin to spinal cord membranes in the presence or absence of capsaicin (10 µM) or PNV (1.5–150 µg/ml). Each column represents the mean± S.E.M of 5–6 mice (A–D) of 3 experiments carried out in duplicate (E). The asterisks denote the significance levels. *P<0.05, ***P<0.001, compared with the vehicle group (A–D: Student's t-test, E: one-way ANOVA followed by the Student-Newman-Keuls test).
Figure 8.
PNV effect on functional TRPV1 channels.
Normalised levels of fluorescence over time from HEK293 cells transfected with rat TRPV1 cDNA and stained with the fluorescent Ca2+ probe fluo-4/AM. Capsaicin (A) or PNV (C) were applied in 20 s pulses as indicated by the horizontal bars above the graphs. No Ca2+ transient signals were observed for capsaicin or PNV addition in non-transfected cells (insets in A and C). Dose-response curves for capsaicin (B) or PNV (D) on transient calcium signals. When applied to cells twice, both capsaicin (200 nM) (E) and PNV (1 µg/mL) (G) elicited Ca2+ transient signals with similar amplitudes. Such response was totally abolished by pre-incubation (300 s) with 10 µM SB366791 (F and H). Points denote mean±S.E.M. of representative plates (10–30 cells, 3 independent experiments).
Figure 9.
The role of TRPV1-expressing sensory fibres or tackykinin NK1 or NK2 receptors in PNV-induced nociception.
The effect of TRPV1-positive sensory fibre ablation with a systemic injection of resinferatoxin (50 mg/kg, A) or the co-administration of the selective antagonists of tackykinin (B) NK1 (SR143033, 0.2 nmol/paw) or NK2 (SR48968, 20 nmol/paw) receptors on PNV-induced (3 µg/paw, i.pl.) nociception in mice. Each column represents the mean±S.E.M of 5–7 mice. The asterisks denote the significance levels. ***P<0.001 in comparison to the vehicle plus vehicle group (A), ###P<0.001 difference in comparison to the PNV plus vehicle treated group (A), *P<0.05, **P<0.01, in comparison to the vehicle plus PNV treated group (B) (one-way ANOVA followed by the Student-Newman-Keuls test).