Role for NGF in augmented sympathetic nerve response to activation of mechanically and metabolically sensitive muscle afferents in rats with femoral artery occlusion

Abstract

Arterial blood pressure and heart rate responses to static contraction of the hindlimb muscles are greater in rats whose femoral arteries were previously ligated than in control rats. Also, the prior findings demonstrate that nerve growth factor (NGF) is increased in sensory neurons-dorsal root ganglion (DRG) neurons of occluded rats. However, the role for endogenous NGF in engagement of the augmented sympathetic and pressor responses to stimulation of mechanically and/or metabolically sensitive muscle afferent nerves during static contraction after femoral artery ligation has not been specifically determined. In the present study, both afferent nerves and either of them were activated by muscle contraction, passive tendon stretch, and arterial injection of lactic acid into the hindlimb muscles. Data showed that femoral occlusion-augmented blood pressure response to contraction was significantly attenuated by a prior administration of the NGF antibody (NGF-Ab) into the hindlimb muscles. The effects of NGF neutralization were not seen when the sympathetic nerve and pressor responses were evoked by stimulation of mechanically sensitive muscle afferent nerves with tendon stretch in occluded rats. In addition, chemically sensitive muscle afferent nerves were stimulated by lactic acid injected into arterial blood supply of the hindlimb muscles after the prior NGF-Ab, demonstrating that the reflex muscle responses to lactic acid were significantly attenuated. The results of this study further showed that NGF-Ab attenuated an increase in acid-sensing ion channel subtype 3 (ASIC3) of DRG in occluded rats. Moreover, immunohistochemistry was employed to examine the number of C-fiber and A-fiber DRG neurons. The data showed that distribution of DRG neurons with different thin fiber phenotypes was not notably altered when NGF was infused into the hindlimb muscles. However, NGF increased expression of ASIC3 in DRG neurons with C-fiber but not A-fiber. Overall, these data suggest that 1) NGF is amplified in sensory nerves of occluded rats and contributes to augmented reflex sympathetic and blood pressure responses evoked by stimulation of chemically, but not mechanically, sensitive muscle afferent nerves and 2) NGF likely plays a role in modulating the muscle metaboreflex via enhancement of ASIC3 expression in C-fiber of DRG neurons.

Fig. 1.

Effects of blocking nerve growth factor (NGF) on the muscle pressor reflex. Muscle contraction was evoked by electrical stimulation of the L4 and L5 ventral roots. Mean arterial pressure (MAP) and heart rate (HR) to static muscle contraction were examined in control rats and rats with 24 h of femoral artery ligation. A: raw data showing that arterial occlusion increased arterial blood pressure (AP) and HR responses to contraction. Prior injection of NGF antibody (10 μg) into the hindlimb muscles attenuated enhancement of the pressor response induced by contraction in occluded rats. B and C: average data for the responses of MAP and HR. *P < 0.05, vs. control and NGF-Ab plus occlusion. There was no significant difference in MAP and HR responses in control rats and occluded rats with NGF antibody injection. D: similar muscle tension development was seen in three groups.

Fig. 2.

Effects of femoral artery occlusion on the sympathetic and pressor responses evoked by stimulation of mechanically sensitive muscle afferent nerves. Responses of renal sympathetic nerve activity (RSNA) and MAP to muscle stretch were observed in control rats and rats whose femoral artery was ligated for 6 (A), 24 (B), and 72 h (C) before the experiments. Here 250 and 500 g of muscle tension was loaded to evoke passive tendon stretch. *P < 0.05, vs. control.

Fig. 3.

Effects of neutralizing NGF on sympathetic and pressor responses evoked by activation of the muscle mechanoreflex. A (original traces) and B (averaged data): RSNA, AP, and HR were examined when muscle stretch was induced by 500 g of tension in control rats and in rats with 24 h of femoral artery occlusion. Significant differences were observed in RSNA and MAP responses to muscle stretch between control and occluded groups. In addition, 24 h of femoral artery ligation was performed on six rats who received NGF-Ab injection. NGF-Ab failed to attenuate occlusion-enhanced responses. Similar time-tension index (TTI) was seen in three groups.

Fig. 4.

Effects of neutralizing NGF on sympathetic and pressor responses evoked by stimulation of muscle chemically sensitive afferent nerves. A (original traces) and B (averaged data) show changes in RSNA and MAP in response to arterial injection of lactic acid (4 μmol/kg) in control limbs and occluded limbs (24 h) with and without NGF-Ab administered previously into the hindlimb muscles. *P < 0.05 vs. control. †P < 0.05 vs. occlusion without NGF-Ab. There were no significant differences in RSNA and MAP responses in control and occlusion plus NGF-Ab.

Fig. 5.

Effects of NGF on expression of ASIC3. A: dual representative bands of ASIC3 expression for control limbs and occluded limbs without and with NGF-Ab infusion. Beta-actin was used as control for equal loading of protein. B: average data. The optical density was expressed in arbitrary units normalized against a control sample. The number of limbs = 5 for each group. *P < 0.05 vs. control. †P < 0.05 vs. without NGF-Ab. C and D: dual representative bands of ASIC3 expression and average data showing that ASIC3 was increased in NGF infusion experiment compared with control. The number of limbs = 6 for each group. *P < 0.05 vs. control. One-way ANOVA was performed to compare variables for ASIC3 optical density in those experiments (B and D).

Fig. 6.

Colocalization of ASIC3 and peripherin. Fluorescence immunohistochemistry was employed to examine double labeling of ASIC3 and peripherin. In this experiment, peripherin was used to label DRG neurons that project C-fiber. A: representative photomicrographs show ASIC3 and peripherin staining in DRG neurons of a control limb (top panel) and a limb with NGF infusion (bottom panel). Arrows indicate representative cells positive for both ASIC3 and peripherin after they were merged. The number of double labeling DRG neurons is greater in infused limbs than that in control limbs. Scale bar = 50 μm. B: average data for colocalization of ASIC3 and peripherin, showing that the percentage of double labeling DRG neurons for ASIC3 and peripherin is greater in NGF infusion limbs than that in control limbs. *P < 0.05 vs. control (one-way ANOVA). No differences were observed in the number of peripherin positive DRG neurons in control and NGF infusion groups.

Fig. 7.

Colocalization of ASIC3 and NF200. Dual fluorescence immunohistochemistry was employed to examine labeling of ASIC3 and NF200. Note that NF200 was used to identify A-fiber of DRG neurons. A: photomicrographs are representative to illustrate staining of ASIC3 and NF200 in DRG neurons of a control limb (top panel) and a limb infused with NGF (bottom panel). Arrows indicate examples for merged ASIC3 and NF200 positive cells. There were no differences in the number of double staining of ASIC3 and NF200 in DRG neurons of control and infused groups. Scale bar = 50 μm. B: average data for colocalization of ASIC3 and NF-200, showing that there are no differences observed in the number of double staining for ASIC3 and NF200 in DRG neurons of control and NGF infusion groups. Note that no differences were observed in the number of NF200 positive DRG neurons in control and NGF infusion groups. One-way ANOVA was performed to compare variables for percentages of double-labeled neurons for ASIC3 and NF200.