Mechanism of heat treatment on exercise pressor reflex in hindlimb ischemia-reperfusion: Does the temperature gradient matter?

Figures

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  Fig. 1.. Heat Treatment (HT) on blood pressure (BP) and heart rate (HR) responses following static muscle contraction. The BP and HR responses were elicited by ~30s static muscle contraction. Peak mean arterial pressure (MAP) and HR were recorded. (A). The MAP responses in IR was higher than sham group and two HT groups. **: between groups: p < 0.01; *: between groups: p < 0.05. (B). No significant differences among groups regarding HR responses to static muscle contraction; (C). The muscle tension during the muscle contraction was higher in IR + HT1.5 °C than IR group. No significant differences was observed in the muscle tension among other groups. **: between groups: p < 0.01. Grey bar: sham group; orange bar: ischemia-reperufsion (IR) group; blue bar: IR group with HT by increasing muscle temperature by ~1.5 °C (IR + HT 1.5 °C); green bar: IR group with HT by increasing muscle temperature by ~3 °C (IR + HT 3 °C). Data was presented as mean ± standard deviation (SD). The numbers in the brackets below the group names indicate the sampe size in each group. The symbols in each data bar indicate the individual values of each data. The BP, HR, and muscle tension responses were collected…

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  Fig. 2.. Heat Treatment (HT) on P2X-mediated blood pressure (BP) and heart rate (HR) responses. The BP and HR responses were elicited by arterial injection of α,β-Me-ATP (0.125 mM). Peak mean arterial pressure (MAP) and HR were recorded. (A). The MAP responses in IR was higher than sham group and two HT groups. *: between groups: p < 0.05. (B). No significant differences among groups regarding HR responses to static muscle contraction. Blue dots: individual data values during pre-α,β-Me-ATP injection control session (saline); purple triangles: individual data values during α,β-Me-ATP injection session; green squares: individual data values during post-α,β-Me-ATP injection recovery session (saline). Data was presented as mean ± standard deviation (SD). The numbers in the brackets below the group names indicate the sampe size in each group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3.. Heat Treatment (HT) on P2X3 protein expression in muscle afferent dorsal root ganglia (DRG). Lumbar 4–6 (L4–6) muscle afferent DRGs were dissected from sham, IR, IR + HT1.5 °C and IR + HT3°C groups. Western blot detected the protein expression of P2X3 (rabbit anti-P2X3 antibody, 1:500, Alomone) in the protein extraction from L4–6 DRG. The optical density was normalized by the β-actin (mouse anti-β-actin, 1:1000, Sigma Aldrich) protein expression in the respective samples. One-way ANOVA with Fisher’s least significant difference test was applied to analyze the difference among the groups. Data bars from left to right: sham, IR, IR + HT1.5 °C and IR + HT°C groups. Compared with sham (n = 11), the P2X3 protein expression was higher in L4–6 DRG of IR rats (n = 11). Both the HT protocols suppressed the P2X3 expression in IR rats. The molecular band indicates that P2X3 was detected near the band of 60 kDa (molecular weight ~ 66 kDa), and β-actin was detected near the band of 40 kDa (molecular weight ~ 42 kDa). Data was presented as mean ± standard deviation (SD). The numbers in the data bar indicate the sample size of each group.**: between groups: p < 0.01; *: between groups: p < 0.05.

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  Fig. 4.. Verification of P2X-mediated Ca2+ entry in isolated muscle afferent afferent dorsal root ganglia (DRG) neurons. In a separate group of sham rats, we tested if the P2X activation and blockage altered the Ca2+ entry of the isolated muscle afferent DRG neurons. (A) Identification of muscle afferent DRG neurons during calcium imaging study. Pictures were taken with the black-and-white colored camera under the microscope. Muscle afferent neurons were isolated from the L4–6 DRG dissected from sham and IR rats. Experiments were performed in neurons that detected both 380 nm (F380, upper panel) and 550 nm (F550, lower panel) wavelengths, indicating those neurons were positive with DiI injected to the gastrocnemius. (B) Data shows that the A317491 (10 μM), a specific P2X2/P2X3 and P2X3 antagonist, significantly reduced the Ca2+ entry activated by α, β-Me-ATP (30 μM), a P2X antagonist. Green bars indicate the application of α, β-Me-ATP (30 μM) and the blue bar indicates the co-administration of α, β-Me-ATP (30 μM) and A317491 (10 μM). Of note, the second green bar indicates the post-washout activation of P2X in the muscle afferent DRG neurons. Data was presented as mean ± standard…

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  Fig. 5.. Heat Treatment (HT) on P2X-mediated Ca2+ entry in isolated muscle afferent DRG neurons. (D). Representative traces of the calcium imaging experiment in isolated muscle afferent DRG neurons from studied groups of animals before and after the administration of α, β-Me-ATP (30 μM). Red line indicated the Ca2+ entry induced by α, β-Me-ATP (30 μM) in muscle afferent DRG neuron of sham rats; Orange line indicates Ca2+ entry induced by α, β-Me-ATP (30 μM) in muscle afferent DRG neuron of IR rats; Green line indicates the Ca2+ entry induced by α, β-Me-ATP (30 μM) in muscle afferent DRG neuron of IR + HT1.5 °C rats; Purple line indicates Ca2+ entry induced by IR + HT3°C in muscle afferent DRG neuron of IR + HT3°C rats. (B). The red bar indicates the sham group, the orange bar indicates the IR group, the green bar indicates the IR + HT1.5 °C group, and the purple bar indicates the IR + HT3°C group. Compared with sham (n = 13), the Ca2+ entry to α, β-Me-ATP (30 μM) was higher in IR group (n = 12). Both the HT protocols suppressed the enhanced IR-induced Ca2+ entry to α, β-Me-ATP (30 μM). Data was presented as mean ± standard deviation (SD). The round dots in the figure bar indicate…