The values for the weight of both whole body and unilateral triceps surae muscles and the relative weight of the muscles expressed as percent body weight are shown for young males (n = 6), young females (n = 5), aged males (n = 5), and aged females (n = 6) in Table 1. Although males and females differed by nearly two-fold in body weight and muscle weight, relative triceps surae muscle weight per body weight was similar and did not differ between males and females in the young adult group, with a median of 0.58% (range: 0.56–0.60%) in males and 0.59% (0.57–0.64%) in females. In contrast, a significant (P < 0.0001) reduction of 55% and 61%, respectively, to 0.32% (0.24–0.39%) and 0.36% (0.35–0.39%) was observed in aged males and females compared with young males and young females, respectively (Table 1). Aged males exhibited greater individual differences in relative muscle weight compared with the other three groups.
Effect of LST transection on TF. Tetanic contractile force was reduced after transection of the lumbar sympathetic trunk in all animals. Figure 1A shows an example of tetanic contraction of the triceps surae muscles in an aged rat under control conditions with intact and transected LST. The lower panel shows the superimposed contraction curves for two conditions, which were averaged over 30 contractions. The peak amplitude of the TF was slightly reduced by the LST transection. Figure 1B summarizes the results of the percent changes in TF amplitude resulting from LST transection in 11 young adult and 11 aged rats. In the young adult and aged groups, the amplitude of TF was significantly reduced after cutting the LST compared with the control condition with intact LST (P < 0.01, by paired t-test). However, the degree of decrease was 6.2% in the aged group, which was half of that in the young adult group (12.9%). There was a significant difference between values in the young and aged groups (P = 0.02, unpaired t-test) (Fig. 1B).
Next, to examine sex differences, a subgroup analysis was performed in four groups, with each value in the young adult and aged group divided by sex. The magnitude of TF reduction in young males, aged males, young females, and aged females was 15.7%, 5.8%, 11.0%, and 8.5%, respectively (Fig. 1C) and there was a significant difference by one-way factorial ANOVA (P = 0.04). The post-test showed a significant difference between the young and aged male groups (P = 0.02), but not between the young and aged female groups (P = 0.9). Because the individual variation of the relative muscle weight was large in aged males, as described above, we examined the correlation between relative muscle weight and the rate of TF reduction by LST transection in the aged male group and observed a strong correlation (r2 = 0.86, P = 0.02) between these two parameters (Fig. 1D).
Effect of LST stimulation on TF. Stimulation of the lumbar sympathetic trunk could increase tetanic contractile force. Figure 2A shows TF recordings with and without stimulation of the peripheral end of the transected LST at a frequency of 10 Hz in an aged rat. TF increased slightly with LST stimulation (solid line) compared to that without (broken line). The effect of LST stimulation was dependent upon the frequency of stimulation (Fig. 2B). In young adult rats, TF was significantly increased at 5 Hz to 20 Hz compared to the pre-stimulus control TF (P < 0.05, by paired t-test), which is consistent with the previous report [20]. In contrast, in aged rats, there were no significant changes at a frequency of 5 Hz (P = 0.19); however, a significant increase was observed at frequencies of 10 Hz and 20 Hz (P < 0.05). The increase in TF in response to LST stimulation at 5 Hz, 10 Hz, and 20 Hz was 4.1%, 3.8%, and 5.8% in the young group and 0.9%, 4.6%, and 9.9% in the aged group, respectively. A two-way ANOVA analysis indicated that, although there was a significant difference in the primary effect of stimulus frequency, there was no significant difference in the effect of aging or interaction. The median of the response to 5 Hz LST stimulation was smaller in aged rats compared with that in young rats; however, the individual variation was large and the difference was not statistically significant.
Finally, a one-way factorial ANOVA analysis of TF responses to 5 Hz, 10 Hz, and 20 Hz LST stimuli, respectively, in the four groups (young males, aged males, young females, and aged females) revealed no significant differences at any stimulus frequency (Supplementary Figure S1A).
Effect of LST stimulation on muscle tonus. Lumbar sympathetic trunk stimulation increased resting muscle tonus. LST stimulation was initiated 1 minute before the onset of tibial nerve stimulation. Therefore, the effect of LST stimulation alone could be observed, independent of motor nerve excitation. Figure 3A shows that LST stimulation alone increased muscle tonus in an aged rat. This increase in tension of more than 20 mg was observed in 7 of 11 rats in the aged group and in only 2 of 11 rats in the young adult group.
The effect of LST stimulation on increasing muscle tonus was dependent upon the frequency of stimulation. In young rats, stimulation at 10 Hz and 20 Hz, but not 5 Hz, produced significant responses (P < 0.05). In contrast, in aged rats, stimulation at frequencies of 5 Hz–20 Hz were effective at producing significant increases in muscle tonus (P < 0.05). The increase in muscle tonus in response to LST stimulation in the aged group was 17 mg, 30 mg, and 33 mg at 5 Hz, 10 Hz, and 20 Hz, respectively, and was 4- to 8-times greater compared with that of the young group, which was 4 mg, 5 mg, and 4 mg, respectively (Fig. 3B). Analysis by a two-way ANOVA revealed a significant difference in the main effect of aging (P = 0.03). Changes in muscle tonus in response to LST stimulation at frequencies of 5 Hz, 10 Hz, and 20 Hz showed similar trends in males and females of the same age group (Supplementary Figure S1B).
LST stimulation at 5–20 Hz resulted in an increase in blood pressure in all rats, regardless of age or sex. The magnitude of the blood pressure increase at 5 Hz, 10 Hz, and 20 Hz stimulation was 8 mmHg, 13 mmHg, and 14 mmHg in the aged group, respectively, which was significantly lower by approximately 30–40% compared with 11 mmHg, 17 mmHg, and 24 mmHg in the young group. There were significant differences between young and aged groups by two-way ANOVA (P < 0.01). These results were similar in both male and female rats (Supplementary Figure S2).
Involvement of adrenergic receptors on LST stimulation-induced modulation of TF and muscle tonus.
Tetanic force. Alpha or beta adrenergic receptor blockade could each reduce the impact of LST stimulation on tetanic contractile force. The effect of adrenergic receptor blockade was examined in cases in which LST stimulation at 10 Hz or 20 Hz increased TF by more than 7%, irrespective of age or sex. Figure 4A shows a typical example of the effect of the α adrenoceptor blocker phentolamine. After phentolamine administration, the magnitude of TF without LST stimulation (dashed line) did not change compared with that before phentolamine administration; however, TF augmentation resulting from LST stimulation (difference between dashed and solid lines) observed before phentolamine injection was somewhat reduced after phentolamine. When summarized for five cases, the LST stimulation-induced modulation of TF was significantly (P < 0.05) attenuated to approximately 40% of the control response before phentolamine administration (Fig. 4B, dark column), without significant change in basal TF values. Next, the effect of the β receptor blocker propranolol was determined in a different set of rats. The propranolol also significantly reduced the LST stimulation-induced modulation of TF to 50% of the control response (P < 0.05) (Fig. 4B, light-colored column). However, after propranolol administration, the basal TF values in the absence of LST stimulation were slightly, but significantly, reduced compared with that before propranolol administration (91% of original). There were individual differences in the effect of each α and β blocker as indicated by the dots in the graphs. When a majority of the response remained with either blocker, a second blocker was administered. Then, the LST-induced increase in TF completely disappeared (4 animals, 2 with phentolamine followed by propranolol and 2 with phentolamine followed by propranolol). These results indicate that both α- and β-receptors are involved in the LST stimulation-induced potentiation of the TF. These results were somewhat similar to a previous report in rabbits, in which the potentiation of jaw muscle twitch contraction resulting from cervical sympathetic trunk stimulation was minimally affected by propranolol, but was attenuated with a subsequent injection with phentolamine [33].
Muscle tonus. Alpha adrenergic receptor blockade, but not beta receptor blockade, eliminated the impact of LST stimulation on muscle tonus. The effect of adrenergic receptor blockers was examined in cases in which LST stimulation (10 Hz or 20 Hz) increased muscle tonus by more than 20 mg. Figure 4C shows a typical example of the effect of phentolamine. Following phentolamine administration, increased response of the muscle tonus resulting from LST stimulation was nearly abolished. To summarize the four cases, the magnitude of the LST stimulation-induced muscle tonus increase was significantly (P < 0.05) ameliorated to approximately 28% of the control response before phentolamine (Fig. 4D, dark column). In contrast, administration of propranolol had no significant effect on the increased muscle tonus response (Fig. 4D, light-colored column). The LST stimulation-induced increase in muscle tonus was minimally affected by administration of the muscle relaxant vecuronium, which was evaluated in 3 rats. These results indicate that LST stimulation induced an increase in muscle tonus, independent of motor nerve activation, via α-receptors, but not β-receptors.