Amplified P2X₃ pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia–reperfusion

Figures

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  FIGURE 1. Expression of P2X3 in L4–6 DRGs. Western blot analysis showing that, compared with sham rats, there was a significant increase in P2X3 protein expression in L4–6 DRGs of IR rats. Data are presented as means ± SD. Numbers in parentheses represent the sample size of each group. **P < 0.01 between IR rats and sham control rats.

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  FIGURE 2. Effect of A‐314791 and siRNA knockdown on P2X3 currents in muscle DRG neurons. Data presented as means ± SD. Numbers in parentheses represent the number of neurons with P2X currents detected. (a) The transient P2X current in the muscle DRG neuron was significantly reduced by A‐317491 (P < 0.05). The decreased transient P2X currents was recovered after the wash‐out period (P < 0.05). *Between groups, P < 0.05. (b) Sustained P2X current in the muscle DRG neuron was significantly reduced by A‐317491 (P < 0.05). The decreased sustained P2X current was recovered after the wash‐out period (P < 0.05). *Between groups as indicated, P < 0.05. (c) Compared with the scramble control, the number and percentage of DRG neurons with P2X currents detected were significantly reduced in muscle afferent DRG neurons in P2X3 siRNA knockdown group (P < 0.05). *Between scramble group and siRNA knockdown group, P < 0.05.

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  FIGURE 3. IR increased P2X currents in IB4‐positive muscle DRG neurons. Data presented as means ± SD in (b–d). (a) Co‐localization of DiI (red) identified as hindlimb muscle innervation and IB4‐positive (green) in DRG neurons for the whole cell patch clamp. (b, c) Data were analysed by chi‐square test. The number and percentage of DRG neurons with P2X currents detected were significantly higher in IB4‐positive DRG neurons than in IB4‐negative DRG neurons. **P < 0.01, IB4‐positive neurons versus IB4‐negative neurons. The distribution of P2X current, namely, transient and sustained currents, in muscle DRG neurons was not different between control rats and IR rats (P > 0.05). Numbers in parentheses represent the number of neurons with P2X currents/total numbers of the neurons evaluated. (d) The amplitude of P2X currents in muscle DRG neurons was significantly higher in IR rats than in sham rats. *P < 0.05, between IR and sham control.

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  FIGURE 4. MAP and HR responses to α,β‐meATP in sham and IR. Data presented as means ± SD. Numbers in parentheses represent the sample size of each group. (a) Compared with sham rats, MAP response to α,β‐meATP was exaggerated in IR rats (P < 0.05). (b) No significant difference was observed in HR response to α,β‐meATP between sham rats and IR rats (P > 0.05).

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  FIGURE 5. Effect of A‐317491 on MAP response to static muscle contraction in IR. Data presented as means ± SD. White bars indicate IR rats with vehicle control (DMSO) treatment and grey bars indicate IR rats with the vehicle control + A‐317491 (DMSO+A‐317491) treatment. Numbers in parentheses represent the sample size of each group. Pre‐injection: before the injection of DMSO or DMSO+A‐317491. Post‐injection: immediately after completing the injection of DMSO or DMSO+A‐317491. (a) Compared with the pre‐injection control, DMSO+A‐314791 significantly reduced the MAP response to static muscle contraction in IR rats. Compared with DMSO alone, DMSO+A‐317491 attenuates the MAP response to static muscle contraction in IR rats. *Versus IR with DMSO, P < 0.05. †Versus pre‐injection control, P < 0.05. (b) No significant difference was observed in the HR response among groups (P > 0.05). (c) No significant difference was found in development of the muscle tension among groups (P > 0.05).

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  FIGURE 6. Effect of A‐317491 on MAP response to α,β‐meATP injection in IR. Data presented as means ± SD. White bars indicate IR rats with vehicle control (DMSO) treatment and grey bars indicate IR rats with the vehicle control + A‐317491 (DMSO+A‐317491) treatment. The sample size of each group is represented as numbers in parentheses (a) and as indicated in (b). Pre‐injection: before the injection of DMSO or DMSO+A‐317491. Post‐injection: immediately after completing the injection of DMSO or DMSO+A‐317491. (a) Compared with the pre‐injection control and DMSO injection, A‐314791 significantly reduced the MAP response to α,β‐meATP. *Versus IR group with DMSO, P < 0.05. †Versus pre‐injection control, P < 0.05. (b) No significant difference was found in the HR response among groups (P > 0.05).