Sex Differences in Vagus Nerve Stimulation Effects on Rat Cardiovascular and Immune Systems

Figures

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  FIGURE 1. Experimental design. (A) Experimental timeline. (B) Vagus nerve cuff electrode, connector mount (prototype), and wireless stimulator. (C) Wireless stimulator connected to an awake behaving rat during experiment, along with VNS parameters.

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  FIGURE 2. A sample ECG recording and estimated instantaneous heart rate (HR) from a male rat during VNS. (A) ECG signal and estimated HR before, during, and after a VNS session. (B–D) 1s ECG snippet for baseline (Pre-VNS), stimulation (VNS), and recovery (Post-VNS), respectively, indicating cardiovascular changes associated with the VNS.

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  FIGURE 3. Normalized short-term trends for ECG morphology and HRV time-domain features (mean ± SEM). (A,B) PR reduces in Sham and Treatment rats, and the effect was more pronounced in the Treatment group with longer recovery to the baseline level (p > 0.05). The average effects for the first hour post-VNS (from 0 to + 50min) shows significant differences between Sham and Treatment groups for male and female rats (p < 0.05) without any significant sex differences. (C,D) RR significantly reduces in the Treatment group with almost one-hour recovery time to the baseline level. The average effects for the first 1 h post-VNS (from 0 to +50 min) show significant differences between Sham and Treatment groups without any significant sex differences. (E,F) RMSSD significantly increases by ∼30% in the Treatment group with ∼20 min recovery. The average trend shows significant treatment effect only in male and significant sex difference in Sham groups. *p < 0.05 and #p < 0.01.

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  FIGURE 4. Normalized long-term trends for ECG morphology and HRV time-domain features (mean ± SEM). (A,B) PR slightly reduces in the Treatment group and increases in the Sham group (not significant; p > 0.05) and slowly recovers toward the VNS endpoint (W14). The average effects for VNS_Early (W7–10), VNS_Late (W11–14), and Recovery (W15–18) periods are compared between groups (Treatment vs Sham and male vs female). Male–female differences are shown for three periods. (C,D) The long-term effect of VNS on RR is significantly different between the Treatment and Sham groups even during the first week of recovery (W15). While sex difference was significant only in the Sham group over three phases, Treatment effect remains significant until W15 for both male and female rats and only for female rats after W15. (E,F) There is no significant difference between the Treatment and Sham groups in the long-term effect of VNS on RMSSD. However, when male and female rats were compared from each group, a significant increase in RMSSD was observed in female rats by VNS, during VNS_Early and VNS_Late phases. *p < 0.05 and #p < 0.01.

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  FIGURE 5. Normalized short-term trends for HRV frequency-domain features (mean ± SEM). (A,B) HF/(LF + HF) indicating the HF proportion of total power shows significant increase in the VNS group with relatively slow transition to the baseline value. Male–female comparison shows that the effect is only significant in male rats. (C,D) LF/HF power ratio or sympathovagal balance shows reduction by VNS (not significant) and slow recovery indicating the predominance of parasympathetic modulation. Male–female comparison shows that the effect is only significant in male rats. *p < 0.05 and #p < 0.01.

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  FIGURE 6. Normalized long-term trends for HRV frequency-domain features (mean ± SEM). (A,B) The long-term effect of VNS on HF/(LF + HF) is not significantly different between the Treatment and Sham groups. However, when male and female rats were compared from each group, a significant increase in HF/(LF + HF) was observed in female rats by VNS, during VNS_Early, VNS_Late, and Recovery phases. (C,D) There is no significant difference between the Treatment and Sham groups in long-term effect of VNS on LF/HF. However, when male and female rats were compared from each group, a significant increase in male and reduction in female rats was observed by VNS during all three shown phases. *p < 0.05 and #p < 0.01.

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  FIGURE 7. Normalized short-term trends for HRV non-linear features (mean ± SEM). (A,B) SD1/SD2 significantly increases by VNS and then recovers after about 30–40 min. Male–female comparison revealed that the effect was only significant in male rats. (C,D) Alpha1/Alpha2 slightly decreases by VNS without significant difference compared to the Sham group. Male–female comparison shows that the effect is only significant in male rats. *p < 0.05 and #p < 0.01.

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  FIGURE 8. Normalized long-term trends for HRV non-linear features (mean ± SEM). (A,B) The long-term effect of VNS on SD1/SD2 is not significantly different between Treatment and Sham groups; however, an increasing trend during VNS is obvious in the Treatment group. VNS effects on SD1/SD2 seem to be similar in male and female rats, but a significant effect was only observed in the female group for early VNS and late VNS phases. (C,D) While VNS slightly decreases Alpha1/Alpha2, there is no significant difference between Treatment and Sham groups in the long-term effect. However, when male and female rats were compared from each group, this reduction appears to be significant only in female rats during VNS_Early and VNS_Late phases. *p < 0.05 and #p < 0.01.

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  FIGURE 9. Weekly concentration of plasma cytokines normalized to the baseline during different experimental timepoints (mean ± SEM). (A,B) TNF-α increases by almost four-fold in both Treatment and Sham groups after implantation of the ECG device on W3 with no consistent trend or significant change by the VNS. Male–female comparison does not reveal any significant effects or sex differences during the five independent phases. (C,D) IFN-γ elevates by the VNS in all rats and then quickly recovers and remains unchanged without significant differences between the Treatment and Sham groups. Male–female comparison does not reveal any significant effects or sex differences during the five independent phases (E,F) IL-1β significantly increases in the Treatment group after the first surgery and then recovers within two weeks and remains unchanged without any difference compared to the Sham group. Male–female difference was only found in the Sham group during the VNS_Early and VNS_Late phases. (G,H) IL-6 significantly increases in the Sham group after the first surgery and then sharply falls below the baseline level at a comparable range as Treatment group with no significant difference. Male–female…