Baroreflex responses to limb venous distension in humans

Abstract

The venous distension reflex (VDR) is a pressor response evoked by peripheral venous distension and accompanied by increased muscle sympathetic nerve activity (MSNA). The effects of venous distension on the baroreflex, an important modulator of blood pressure (BP), have not been examined. The purpose of this study was to examine the effect of the VDR on baroreflex sensitivity (BRS). We hypothesized that the VDR will increase the sympathetic BRS (SBRS). Beat-by-beat heart rate (HR), BP, and MSNA were recorded in 16 female and 19 male young healthy subjects. To induce venous distension, normal saline equivalent to 5% of the forearm volume was infused into the veins of the occluded forearm. SBRS was assessed from the relationship between diastolic BP and MSNA during spontaneous BP variations. Cardiovagal BRS (CBRS) was assessed with the sequence technique. Venous distension evoked significant increases in BP and MSNA. Compared with baseline, during the maximal VDR response period, SBRS was significantly increased (−3.1 ± 1.5 to −4.5 ± 1.6 bursts·100 heartbeats⁻¹·mmHg⁻¹, P < 0.01) and CBRS was significantly decreased (16.6 ± 5.4 to 13.8 ± 6.1 ms·mmHg⁻¹, P < 0.01). No sex differences were observed in the effect of the VDR on SBRS or CBRS. These results indicate that in addition to its pressor effect, the VDR altered both SBRS and CBRS. We speculate that these changes in baroreflex function contribute to the modulation of MSNA and BP during limb venous distension.

INTRODUCTION

The distension of peripheral veins has been reported to activate sympathetic efferent activity and increase blood pressure (BP) both in animals (1) and humans (2). Venous distension increased group III and IV afferent nerve discharges in cat limbs (3). Afferent nerve endings in peripheral veins have been reported to sense changes in blood volume and contribute to BP regulation in dogs (4), rabbits (5), and rats (1). Regarding humans, we (2) and others (6) previously reported that venous distension induced by a volume infusion (saline) into the veins of the occluded forearm provoked a significant increase in BP and muscle sympathetic nerve activity (MSNA) in healthy humans. Lidocaine added to the injection fluid to the occluded forearm abolished the MSNA and BP response to the venous distension (7). Therefore, the increase in MSNA and BP elicited by venous distension can be regarded as a sympathoexcitatory reflex (termed venous distension reflex, VDR).

The arterial baroreflex is a well-known and crucial regulator of BP (8–10). The arterial baroreflex regulates heart rate (HR) and sympathetic nerve discharges to peripheral vascular beds (i.e., MSNA) as a negative feedback mechanism that counteracts changes in BP (11). Under resting conditions in healthy individuals, there are close inverse relationships between the HR and systolic blood pressure (SBP) and between the occurrence of MSNA bursts and diastolic blood pressure (DBP) (12, 13). The slopes of these relationships (i.e., baroreflex sensitivity curves) can be used to determine the sensitivity of baroreflex control of HR (i.e., cardiovagal baroreflex sensitivity, CBRS) and sympathetic nerve activity (i.e., sympathetic baroreflex sensitivity, SBRS) and to define their operating points, respectively (14).

Besides the arterial and cardiopulmonary baroreflexes, sympathetic and parasympathetic activities are also modulated by other reflex mechanisms [e.g., exercise pressor reflex (15)]. It is known that varied conditions (e.g., exercise, mental stress) (16, 17) can affect baroreflex function (e.g., CBRS, SBRS, and operating points). The interaction between the baroreflex and other autonomic nerve reflexes (18, 19) may keep the feedback system effective to maintain hemodynamic stability (20). For example, when nerve afferents were activated by the cold pressor test (21) or by metaboreceptor stimulation (e.g., postexercise muscle ischemia) (22–24), these interventions reset the baroreflex curve and the operating point to a higher pressure (upward and rightward shift). Moreover, increased SBRS was observed during either the cold pressor test (21) or metaboreceptor stimulation (22–24) when both MSNA and BP were raised. Elevated SBRS may help to suppress MSNA to some extent and in turn, avoid an exaggerated pressor response.

Previous reports (2, 6, 7, 25–28) show that venous distension induced increases in both BP and MSNA, while HR increased slightly. Because both MSNA and BP increased during venous distension, it might imply that baroreceptor inhibition of MSNA is “overridden” by the VDR or the VDR may reset the baroreflex. Moreover, baroreflex control of HR may also be altered by the VDR as the HR was not decreased despite an increase in BP (2, 7, 26–28). Although the increases in both MSNA and BP could suggest a shift in baroreflex curve, how the VDR affects the slope of the baroreflex curve has not yet been established. Thus, the purpose of this study was to examine the effects of the VDR on baroreflex function. As described in the the introduction, we speculate that venous distension would stimulate group III and IV afferents, which raises MSNA and results in a pressor response. We further speculate that increased SBRS during VDR activation may serve to avoid an exaggerated pressor response as seen during the cold pressor test (21) or metaboreceptor stimulation (22–24). Thus, we hypothesized that the VDR would act to increase the sensitivity of baroreflex control of MSNA in healthy individuals. It is well known that there are sex differences in BP control under varied conditions (29, 30). Whether sex differences are also present in baroreflex function during venous distension is unclear. Thus, in this study, we also examined potential sex differences in BRS during VDR activation.

METHODS

Participants

Thirty-five young healthy subjects (19 males and 16 females; average age, 26.7 ± 3.4 yr) were included in this study. All subjects were free of any diseases and used no medications. The average height and weight were 171.2 ± 9.0 cm and 72.6 ± 12.9 kg, respectively. Subjects were instructed to refrain from alcohol, caffeine, and strenuous exercise for 24 h before the laboratory visit. The study protocol was approved by the Institutional Review Board of the Milton S. Hershey Medical Center (Study 27242) and conformed with the Declaration of Helsinki. The purpose, protocol, and risks were explained to each subject before written informed consent was obtained. Data from a control trial in 14 subjects in one of our prior reports (7, 26) were included in the analysis in this report. However, the baroreflex activity during VDR activation in these 14 subjects in the present study was not published in the prior report (7, 26). The data from the other 21 subjects were not included in prior reports. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Measurements

As described in our earlier reports (2, 26), beat-to-beat HR was obtained from the electrocardiogram (Cardicap 5, Datex-Ohmeda, GE, Healthcare) and beat-to-beat BP was measured via finger photoelectric plethysmography (Finometer, Finapress Medical System, Amsterdam, The Netherlands). Baseline beat-to-beat BP values were verified by the upper arm cuff pressure (SureSigns, VS3, Philips, Philip Medical system). Respiratory frequency was monitored using piezoelectric pneumography and subjects were instructed to avoid holding their breath during the experiment. MSNA was directly recorded from the peroneal nerve using a tungsten microelectrode as described previously (31). Briefly, the microelectrode was inserted percutaneously and adjusted to obtain spontaneous pulse synchronous multiunit bursts of MSNA, which agree with established criteria (32). The nerve signal was amplified, bandpass-filtered (500–5,000 Hz), rectified, and integrated with a time constant of 0.11 s (Iowa Bioengineering, Iowa City, IA). The MSNA signal was audibly and visually monitored throughout the protocol to ensure that the MSNA recording remained stable throughout the study.

Experimental Protocol

The subjects entered the laboratory after voiding and underwent anthropometric measurements. Then, as described in earlier reports (7, 26), forearm volume of the nondominant arm (from elbow to wrist) was assessed by the water displacement method (33). Forearm volume was calculated by subtracting the hand (to wrist) volume from the limb (to elbow) volume. Thereafter, subjects lied in a supine position for instrumentation including placement of a tungsten microelectrode for MSNA recording. An intravenous catheter was inserted in the antecubital fossa of the nondominant arm.

After instrumentation, subjects were asked to rest quietly during an acclimation period before data collection. The acclimation period lasted at least 5 min or until the hemodynamic variables and MSNA were stable. Baseline recordings of ECG, BP, respiratory traces, and MSNA were performed for 6 min. Thereafter, venous distension was induced by the same method as we described previously (7, 26, 28). Briefly, the arm was elevated and was fitted with three occlusion cuffs from the wrist to the elbow, and a fourth cuff was placed on the upper arm. The cuffs were inflated to 250 mmHg, sequentially from the wrist to the upper arm. Then, the three cuffs on the forearm were deflated and removed. Although the upper arm cuff remained inflated, the arm was returned to the horizontal position [we termed this procedure “Wrist to Elbow (WE) occlusion”]. After 4 min of the preinfusion period, normal saline (0.9% concentration) equal to 5% forearm volume was infused in the occluded forearm via the intravenous catheter at an infusion rate of 30 mL/min. Depending on the infused volume (∼40–70 mL), the infusion lasted ∼1.3–2.3 min. The fourth (upper arm) cuff was deflated 5 min after completion of the infusion. Because the peak response of MSNA and BP were observed near the end of the infusion, we defined the last 30 s of the infusion and the first 30 s of the postinfusion period (1 min total) as the VDR period (7, 26, 28) (Fig. 1).

Figure 1.

A: schematic diagram of the experimental protocol. B: representative recordings of muscle sympathetic nerve activity (MSNA), blood pressure (BP), and beat-by-beat heart rate (HR). Time scales of the bottom panel are different from the top panel. Post-Inf, post-infusion; Pre-Inf, pre-infusion; VDR, maximal response period to venous distension; WE, wrist to elbow occlusion.

Data Analysis

Signals were recorded at a sampling frequency of 200 Hz via a dedicated data-acquisition system (MacLab, AD Instruments, Castle Hill, Australia) (34). MSNA bursts on the integrated MSNA tracings were visually identified and confirmed by the burst sound from the audio amplifier. As described in earlier reports (21, 34), MSNA bursts were evaluated by a computer program that identified bursts based upon fixed criteria, including an appropriate latency following the R-wave of the electrocardiogram, and having a signal-to-noise ratio of at least 2:1, and were verified by visual review of the recording. Integrated MSNA bursts were normalized by assigning a value of 100 to the mean amplitude of the top 10% largest bursts among all bursts during the baseline period to reduce variability attributed to factors including needle position and signal amplification (34). Total MSNA activity for each cardiac cycle was calculated from the burst area of the integrated neurogram. If no MSNA bursts were detected for a particular cardiac cycle, a zero value was assigned for the cardiac cycle. MSNA was reported as the number of bursts per minute (burst frequency, BF), the number of bursts per 100 heartbeats (burst incidence, BI), and total MSNA (units/min), which was calculated as the sum of the burst area of the integrated neurogram on a beat-to-beat basis. Beat-to-beat HR, systolic BP (SBP), diastolic BP (DBP), and total MSNA were obtained continuously from baseline to 5 min after the end of the infusion.

Averaged values of MSNA, BP, and HR were analyzed over the 6-min freely perfused baseline, the last 3 min of the 4-min pre-infusion (Pre-Inf) period, and the VDR period as defined in the protocol (i.e., the mean values obtained during the last 30 s of infusion and the first 30 s of the postinfusion period) as shown in Fig. 1. CBRS and SBRS were calculated for each of these 3 periods.

CBRS was calculated as the slope of the linear relationship between SBP and the cardiac RR interval (RRI) (y-axis: RRI, x-axis: SBP) (11, 35) by the sequence method using Hemolab software (Harald Stauss Scientific, Iowa City, IA) (36). As described in earlier reports (37, 38), sequences of three or more beats, during which SBP and the RRI of the following beats altered in parallel were detected. A least-square linear regression analysis was applied to each sequence and only sequences in which R² > 0.8 were accepted. The average value of the sequences (including both increasing and decreasing SBP sequences) was defined as CBRS (ms/mmHg).

SBRS was assessed from the slope of the linear regression between diastolic BP (DBP) and MSNA (BI, burst area, and total MSNA) with spontaneous BP changes (y-axis: MSNA, x-axis: DBP) as described in earlier reports (39–41). As described in our earlier reports (37, 38), DBP for each cardiac interval was classified into 3 mmHg interval bins. “BI” for a given 3-mmHg DBP bin was expressed as the percentage of cardiac cycles in which a burst occurred for a given DBP bin. For each 3-mmHg DBP bin, the total burst area was calculated and was divided by the number of MSNA bursts occurring within the respective bin. This value presented the mean “burst area” for a given DBP bin. The total burst area within a DBP bin was divided by the number of cardiac cycles that occurred within that interval, which represented the “total MSNA” for a given DBP bin. By using these binned data, the SBRS slopes for the linear relationship between MSNA (BI, burst area, and total MSNA) and DBP were calculated (SBRS-BI, SBRS-burst area, and SBRS-total MSNA). For correlation coefficients (R values) of the regression line ≥ 0.5, the slope of the line was regarded as an acceptable SBRS slope (13). An example of the linear regression between MSNA BI and DBP for a representative subject is shown in Fig. 2.

Figure 2.

Representative curves of baroreflex control of MSNA burst incidence during baseline and the venous distension period in one subject. The slope of the linear regression between MSNA and diastolic blood pressure in the two conditions (baseline and VDR) was calculated to determine baroreflex sensitivity. VDR increased both MSNA and DBP, as indicated by a right and upward shift in the operating point. It is also clear that the slope relating the change in MSNA relative to the change in DBP was more negative during VDR. At baseline, ES was negative, whereas it was positive during VDR period. DBP, diastolic blood pressure; ES, error signal; MSNA, muscle sympathetic nerve activity; T50, the diastolic blood pressure corresponding to burst incidence = 50 on the curve; VDR, venous distension reflex.

SBRS operating points were determined as the points corresponding to the average DBP on the regression lines relating MSNA (BI, total MSNA, and burst area) to DBP in each subject (18, 29, 42). The SBRS-BI “T50” value was calculated as the DBP at which 50% of the cardiac cycles were related to a burst (i.e., DBP corresponding to BI = 50 on the SBRS-BI curve) (41) (see Fig. 2). The SBRS “error signal (ES)” was defined as the difference between T50 and the operating point in each subject (T50 minus average DBP) (29) (see Fig. 2). The differences in variables (HR, BP, MSNA, BRS, T50, and ES) were compared between baseline, pre-infusion (Pre-Inf), and VDR period in all patients.

Sex Difference

Sex differences were observed in the sympathetic responses to varied conditions including chemoreflex stress or exercise (43, 44). Whereas a recent study (6) suggested that there was no sex difference in MSNA responses to venous distension, the effect of sex on baroreflex function during VDR stimulation has not been examined. Although this study was not specifically designed for this purpose, we examined sex differences with data collected from a similar number of male (subject number n = 19) and female (n = 16) subjects.

Statistical Analysis

All statistical analyses were performed using SPSS software (SPSS Science v. 27.0, IBM). Unpaired t tests were used to compare differences in baseline characteristics between males and females. A linear mixed-effects model (38, 45) was used for most of the analyses in this study. First, a linear mixed-effects model was performed to evaluate the effect of the intervention (baseline, Pre-Inf, and VDR) on variables (MAP, HR, MSNA, CBRS, SBRS, T50, and error signal values) in all subjects. Because of earlier reports of a relationship with baroreflex function (46, 47), age and body mass index (BMI) were applied as covariates. Second, to examine sex differences, the effect of the intervention, sex (male or female), and the interaction (intervention × sex) on parameters in the linear mixed-effects model were calculated. Age and BMI were applied as covariates to the linear mixed-effects models. Bonferroni post hoc testing was performed when appropriate. All data were presented as means ± standard deviation (SD). In all analyses, values of P < 0.05 (two-sided) were considered statistically significant.

RESULTS

The baseline characteristics in all subjects and each group are shown in Table 1. BMI and SBP were higher in males than in females. There were no significant differences in age, DBP, MAP, HR, MSNA BF, and BI between males and females in these subjects (Table 1).

Table 1.

Baseline characteristics

	All	Male	Female	P
Age, yr	26.7 ± 3.4	26.3 ± 3.1	27.3 ± 3.7	0.425
BMI, kg/m2	24.7 ± 3.3	26.1 ± 3.4	22.9 ± 2.4	0.003
SBP, mmHg	120.8 ± 18.1	127.1 ± 20.9	113.3 ± 10.4	0.017
DBP, mmHg	67.6 ± 7.3	68.0 ± 7.2	67.0 ± 7.7	0.701
MAP, mmHg	85.5 ± 9.3	87.7 ± 10.7	82.9 ± 6.5	0.124
HR, beats/min	65.2 ± 9.0	64.6 ± 10.4	66.0 ± 7.3	0.658
MSNA BF, bursts/min	23.5 ± 8.8	23.4 ± 8.1	23.6 ± 9.7	0.947
MSNA BI, bursts/100 heartbeats	36.1 ± 13.8	36.4 ± 13.0	35.8 ± 15.0	0.913

Data are presented as means ± SD. BMI, body mass index; DBP, diastolic blood pressure; HR, heart rate; MAP, mean arterial pressure; MSNA, muscle sympathetic nerve activity; MSNA BF, MSNA burst frequency; MSNA BI, MSNA burst incidence; SBP, systolic blood pressure. P (male vs. female), P value of unpaired t tests between males and females. Subject number n = 35 (male, n = 19; female, n = 16). MSNA data were recorded in 34 subjects (male, n = 18; female, n = 16).

Responses of HR, MAP, and MSNA to Venous Distension

MSNA was successfully recorded throughout the trial in 34 subjects (male, n = 18; female, n = 16). Representative recordings of MSNA, HR, and BP during baseline, preinfusion, and VDR period from one subject are shown in Fig. 1. MSNA and BP clearly increased after the forearm infusion (i.e., during the VDR period). Average values of MAP, HR, and MSNA in all subjects are shown in Table 2. MAP, HR, and MSNA in the Pre-Inf period were not statistically different from those during baseline but were significantly increased in the VDR period compared with other periods (MAP, MSNA BF, BI, and total MSNA, P < 0.0001; HR, P = 0.006) (Table 2).

Table 2.

Effects of VDR activation on cardiovascular variables and muscle sympathetic nerve activity

	Base	Pre-Inf	VDR	P (Intervention)
MAP, mmHg	85.5 ± 9.3	88.9 ± 10.0	100.2 ± 13.6*†	<0.0001
HR, beats/min	65.2 ± 9.0	65.5 ± 9.2	68.0 ± 11.5*†	0.006
MSNA BF, bursts/min	23.5 ± 8.8	25.2 ± 8.8	33.5 ± 9.2*†	<0.0001
MSNA BI, bursts/100 heartbeats	36.1 ± 13.8	38.7 ± 14	50.1 ± 16.4	<0.0001
Total MSNA, units/min	449 ± 155	498 ± 159	914 ± 386	<0.0001

Data are presented as means ± SD Base, baseline; BF, burst frequency; BI, burst incidence; HR, heart rate; MAP, mean arterial pressure; MSNA, muscle sympathetic nerve activity; Pre-Inf, pre-infusion; VDR, maximal response period. Age and body mass index are applied as covariates in the linear mixed-effects model. *P < 0.05 compared with baseline; †P < 0.05 compared with Pre-Inf. P (intervention), effect of intervention in the linear mixed-effects model. Subject number n = 35. MSNA data were recorded in 34 subjects.

Average values of MAP, HR, MSNA BF, and MSNA BI in males and females are shown in Fig. 3. The effects of the intervention on all parameters (HR, MAP, BF, and BI) were significant (MAP, MSNA BI, and total MSNA, P < 0.0001; HR, P = 0.006); however, the effects of “sex” on all parameters were not significant (Fig. 3) (all, P > 0.05). The post hoc analysis showed that there are no significant differences in all parameters between males and females in each period (Baseline, Pre-Inf, and VDR, all, P > 0.05) (Fig. 3). The VDR also evoked significant increases in MSNA total activity in males (Baseline, 440 ± 158; Pre-Inf, 492 ± 177; VDR, 855 ± 313 units/min; effect of intervention, P < 0.0001; post hoc: VDR vs. baseline and Pre-Inf, both P < 0.0001) and females (Baseline, 458 ± 156; Pre-Inf, 504 ± 142; VDR, 980 ± 456 units/min; effect of intervention, P < 0.0001; post hoc: VDR vs. baseline and Pre-Inf, both P < 0.0001), respectively.

Figure 3.

Absolute values of cardiovascular variables and muscle sympathetic nerve activity (MSNA) during venous distension in male and female subjects. A: mean arterial pressure (MAP). B: heart rate (HR). C: MSNA burst frequency (BI). D: total MSNA. White bars indicate average values in males and gray bars indicate average values in females. Small circles represent individual values of male subjects. Small triangles represent individual values of female subjects. P values for intervention, sex, and the interaction were from the linear mixed-effects model. Age and BMI were applied as covariates in the linear mixed-effects model. Post hoc: *P < 0.05 compared with baseline; †P < 0.05 compared with Pre-Inf. There was no significant difference in these variables between male and female groups. Base, baseline; BMI, body mass index; Pre-Inf, pre-infusion; VDR, maximal response period.

Responses of Cardiovagal BRS and Sympathetic BRS to Venous Distension

CBRS and SBRS (SBRS-BI, -Total MSNA, and -burst area) during the three periods in all subjects are shown in Table 3. CBRS was successfully calculated in all subjects (subject number n = 35). SBRS-BI and SBRS-Total were successfully calculated in all the 3 periods in 29 subjects (male, n = 15; female, n = 14). The other six subjects were excluded because of a low correlation coefficient (R) value (< 0.5) in one (or more) of the three periods. The mean R values of SBRS in all three periods (Base, Pre-Inf, and VDR) were: 0.93 ± 0.06, 0.88 ± 0.10, 0.90 ± 0.08 for SBRS-BI; and 0.95 ± 0.05, 0.90 ± 0.10, 0.90 ± 0.08 for SBRS-total MSNA, respectively. The number of subjects whose SBRS-burst area was acceptable (R ≥ 0.5) during the baseline, Pre-Inf, and VDR periods was 17 (male, n = 9; female, n = 8), 23 (male, n = 11; female, n = 12), 20 (male, n = 12; female, n = 8), respectively.

Table 3.

Baroreflex sensitivity during venous distension in all subjects

	Base	Pre-Inf	VDR	P (Intervention)
CBRS, ms/mmHg	16.6 ± 5.4	16.5 ± 6.1	13.8 ± 6.1*†	0.0006
SBRS-BI, bursts·100 heartbeats−1·mmHg−1	−3.1 ± 1.5	−3.8 ± 1.8	−4.5 ± 1.6*†	0.0011
SBRS-Total MSNA, units·beats−1·mmHg−1	−0.7 ± 0.4	−1.0 ± 0.5	−1.5 ± 0.6*†	<0.0001
SBRS-Burst area, units·beats−1·mmHg−1	−0.7 ± 0.4	−0.8 ± 0.4	−1.3 ± 0.6*†	<0.0001

Data are presented as means ± SD. Base, baseline; BI, burst incidence; BMI, body mass index; CBRS, cardiovagal baroreflex sensitivity; MSNA, muscle sympathetic nerve activity; Pre-Inf, pre-infusion; SBRS, sympathetic baroreflex sensitivity; VDR, maximal response period. Age and BMI are applied as covariates in the linear mixed-effects model. *P < 0.05 compared with baseline; †P < 0.05 compared with Pre-Inf. P (intervention), effect of intervention in the linear mixed-effects model. Subject number n = 35 for CBRS, n = 29 for SBRS-BI, and SBRS-total MSNA.

With the data of all subjects (Table 3), CBRS during VDR was significantly decreased compared with the baseline and Pre-Inf periods (effect of the intervention, P = 0.0006). SBRS-BI during the VDR was significantly increased compared with the baseline (effect of the intervention, P = 0.0011), and SBRS-total MSNA and SBRS-burst area during VDR were significantly increased compared with the baseline and Pre-Inf periods (effect of the intervention, P < 0.0001). There were no significant differences in the CBRS and all SBRS parameters between the baseline and pre-Inf periods (all, P > 0.05) (Table 3).

CBRS and SBRS (SBRS-BI, -total MSNA, -burst area) during the three periods in males and females are shown in Fig. 4. There was no significant sex difference in CBRS and SBRS (SBRS-BI, -total MSNA, and -burst area) throughout the trial (effect of sex, P > 0.05; effect of the intervention × sex, P > 0.05) (Fig. 4).

Figure 4.

Baroreflex sensitivity during venous distension in male and female subjects. A: cardiovagal baroreflex sensitivity (CBRS). B: sensitivity of baroreflex control of MSNA burst incidence (SBRS-BI). C: sensitivity of baroreflex control of MSNA total activity (SBRS-total MSNA). D: sensitivity of baroreflex control of MSNA burst area (SBRS-burst area). White bars indicate average values in males and gray bars indicate average values in females. Small circles represent individual values of male subjects. Small triangles represent individual values of female subjects. P values for intervention, sex, and the interaction were from the linear mixed-effects model. Age and BMI were applied as covariates in the linear mixed-effects model. Post hoc: *P < 0.05 compared with baseline; †P < 0.05 compared with Pre-Inf. ‡P < 0.05 compared with male group in the same period. Base, baseline; BMI, body mass index; MSNA, muscle sympathetic nerve activity; Pre-Inf, pre-infusion; VDR, maximal response period.

The effects of the VDR on T50 and ES are shown in Table 4. Based on data from all subjects, T50 was significantly increased during VDR compared with Pre-Inf and baseline (effect of the intervention, P < 0.0001), suggesting a rightward shift of the SBRS curve. When the data were analyzed separately for the male and female groups, the effect of sex on T50 was significant (P = 0.019). Baseline T50 in females tended to be lower than in males (P = 0.069), and T50 during Pre-Inf and VDR in females was significantly lower than in males (Pre-Inf, P = 0.026; VDR, P = 0.031) (Table 4). Although the effect of “intervention and sex interaction (intervention × sex)” on T50 was not significant (P = 0.882), with the data of all subjects, ES was significantly increased during VDR compared with baseline and Pre-Inf (effect of the intervention, P < 0.0001), and average ES was positive only during the VDR period (all subjects, 0.6 ± 3.7). No difference was observed in the ES between Pre-Inf and baseline. When the data were sorted into male and female groups, no sex difference was observed for ES (effect of the sex, P = 0.809; effect of the intervention × sex, P = 0.743; sex difference in each period, P > 0.05) (Table 4).

Table 4.

Effects of VDR activation on T50 and error signal

	Base	Pre-Inf	VDR
T50, mmHg
all	59.1 ± 13.0	63.2 ± 13.1	77.8 ± 13.7*†
P (factor)	<0.0001 (Intervention)	0.019 (sex)	0.882 (Intervention × sex)
ES, mmHg
all	−5.4 ± 7.3	−3.9 ± 7.1	0.6 ± 3.7*†
P (factor)	<0.0001 (Intervention)	0.809 (sex)	0.743 (Intervention × sex)
T50, mmHg
Male	63.3 ± 12.5	68.3 ± 14.1	82.8 ± 8.3
Female	54.6 ± 12.4	57.7 ± 9.7	72.5 ± 16.5
P (male vs. female)	0.069	0.026	0.031
ES, mmHg
Male	−5.6 ± 6.6	−3.1 ± 8.8	0.7 ± 4.9
Female	−5.2 ± 8.2	−4.7 ± 4.8	0.5 ± 2.1
P (male vs. female)	0.862	0.514	0.932

Data are presented as means ± SD. Base, baseline; BMI, body mass index; Pre-Inf, pre-infusion; T50, the diastolic blood pressure corresponding to burst incidence = 50 on the curve; VDR, maximal response period. *P < 0.05 compared with baseline; †P < 0.05 compared with Pre-Inf. P (factor), effect of factors (intervention, sex, and intervention-sex interaction) in the linear mixed-effects model. Age and BMI are applied as covariates in the linear mixed-effects model. Post hoc: P values for male vs. female during each period are indicated in the table. Subject number n = 29 (male, n = 15: female, n = 14).

DISCUSSION

The main findings of this study are that 1) VDR activation significantly increased SBRS with a rightward and upward shift (resetting) of the curve of DBP versus MSNA and was accompanied by a rightward shift (to higher pressure) of the T50 value, which is defined as the DBP level at which 50% of the heartbeats are accompanied by an MSNA burst (29, 41); 2) VDR activation significantly decreased CBRS; and 3) the effects of VDR on BRS were similar in males and females. These findings support our hypothesis that the VDR increases the gain of baroreflex control of MSNA in young healthy males and females.

BP, HR, and MSNA Responses to VDR Activation

In the present study, MAP and all MSNA parameters (BF, BI, and total MSNA) were significantly increased during VDR activation, which is consistent with previous reports (2, 6, 7, 25–28). In addition, in the present study, in all subjects, the HR was slightly but significantly increased by the VDR. This is consistent with some of the previous reports (2, 25), while others (6, 7, 26–28) showed that HR during VDR activation was slightly higher than baseline, but this did not reach statistical significance. The difference in the significance of these observations could be due to the differences related to subjects or the subject number. Of note, the present study (subject number n = 35) was much larger than the earlier studies cited.

In the present study, the responses of MSNA and BP to the VDR in females were not different from those in males. These data suggest that there were no sex differences in the responses of MSNA and BP to the VDR. The lack of sex differences in MSNA responses to the VDR was consistent with a recent study (6). However, in that report (6), the BP responses to VDR activation in females were lower than those in males. We speculate that differences in subjects studied could have contributed to the differences in these observations. It should be noted that in that report (6) only women in the early follicular phase of the menstrual cycle (first 5 days) were studied, whereas the present study included women in other phases of the menstrual cycle (2 females were in the early follicular phase, 6 females were in the late follicular phase, and 8 females were in the luteal phase).

Effects of the VDR on SBRS and CBRS

In the present study, we found that the VDR increased SBRS (i.e., the slope of the curve of DBP vs. MSNA) and shifted the baroreflex curve rightward (i.e., to a higher-pressure range). As shown in a representative example in Fig. 2, the rightward and upward resetting of the baroreflex curve will induce an increase in MSNA when BP is at a given level (20). In the present study, both the operating points [i.e., the point corresponding to the average DBP on the baroreflex curve (18, 29, 42)] and T50 i.e., the DBP corresponding to MSNA BI = 50 on the SBRS-BI curve (41)] were determined from the baroreflex curve (see Fig. 2). The error signal (ES) was the difference between T50 and the operating DBP (i.e., T50 − average DBP) (29). During baseline, ES was negative (i.e., T50 < average DBP), which indicates the baroreceptors were loaded when the BP was around the operating point (Fig. 2). Thus, MSNA would be suppressed to some extent under this condition. During the VDR period, the ES value became positive (i.e., T50 > operating point). This suggests that the baroreceptors were unloaded around the operating point. Thus, MSNA would be activated under this condition (Fig. 2). Importantly, under this condition, with increased SBRS (i.e., steeper slope), the baroreflex would evoke greater MSNA activation than that with a lower SBRS. Thus, both resetting of the baroreflex curve and increased reflex sensitivity would contribute to MSNA activation during limb venous distension. On the other hand, the VDR decreased the CBRS slope. Decreased CBRS could cause an attenuation of the ability to buffer a BP increase (48, 49). Therefore, we speculate that altered CBRS and SBRS as well as resetting of the baroreflex curves could contribute to the increase of MSNA during VDR activation.

Within males or females, the change in SBRS-BI by VDR activation was less significant compared with that of SBRS-total MSNA (Fig. 4). Total MSNA reflects both burst frequency and strength and has been regarded as a better index of MSNA than assessing frequency and strength separately (50, 51). Thus, it is not surprising that SBRS-total MSNA was a more sensitive parameter compared with SBRS-BI. In fact, in several earlier studies, significant changes in SBRS-total MSNA without a change in SBRS-BI by the intervention were reported (38, 52, 53). Meanwhile, it was suggested that the number of bursts (gating effect) and the amplitude of the bursts are regulated differently in the central nervous system (39). Thus, we speculate that the effect of VDR on SBRS might be dominant in the regulation of MSNA burst amplitude compared with the regulation of frequency.

The reflex response to venous distension, i.e., the VDR, is thought to be induced via vessel wall deformation that causes afferent activation of group III and IV nerves innervating the adventitia of veins (6, 26). Group III and IV afferents also play a role in the exercise pressor reflex (EPR) (24). The EPR consists of a mechanoreflex (group III and IV nerves) and a metaboreflex (predominantly group IV nerves) component (24). Mechanoreflex (deformation of exercising muscle) and metaboreflex (metabolic by-products of exercise) activation are reported to shift (reset) the baroreflex curve (16, 22, 54). Mechanoreflex activation is suggested to inhibit vagal outflow and to decrease CBRS (48, 49), whereas metaboreflex activation is suggested to increase SBRS (22, 23). In view of these earlier findings, we speculate that activation of group III and IV afferents by venous distension alone alters BRS. On the other hand, we could not confirm whether group III or IV afferents activated by venous distension were the same as those activated by exercise. We recently found that the VDR could be normally activated even during metaboreflex activation evoked by postexercise muscle ischemia (28). This suggests that, in agreement with an earlier report (3), the afferents stimulated by venous distension and those stimulated by the metaboreflex might not be the same. Even so, the effects of activation of group III and IV afferents on BRS might be similar.

Sex Difference in CBRS and SBRS in Response to the VDR

In the present study, responses in SBRS-BI or CBRS to VDR activation were not significantly different between males and females. However, there was a tendency (P = 0.073) for an interaction between sex and the intervention in SBRS-BI (Fig. 4). Thus, we speculate that when controlling for the phase of the menstrual cycle in females, a sex difference in SBRS-BI responses to venous distension might be observed. Thus, future studies with a larger number of females classified by their menstrual cycle phase are needed.

The baseline T50 values in males tended to be higher than in females at baseline (P = 0.069) and T50 in males remained higher than in females during Pre-Inf and VDR (effect of sex, P = 0.019; Pre-Inf, P = 0.026; VDR, P = 0.031). The T50 value was suggested to be affected by sex hormones (e.g., estrogen) (29, 55). In previous studies, average T50 values in young females were lower than in young males (29) and postmenopausal females (55). Accordingly, it is not surprising that T50 values in females were lower than in males in the present study. Meanwhile, in the present study, T50 and ES were significantly increased during VDR compared with other periods in both males and females, and the degree of changes did not differ by sex. From these results, it is speculated that the changes in CBRS and SBRS, including T50 and ES, in response to VDR activation were not different between males and females. It has been reported that MSNA and BRS values in females vary by their hormone (i.e., estrogen and progesterone) level (56). This points to the need to consider the role of the stage of the menstrual cycle. However, because of the small sample size in our study, it was difficult to compare the values by the phase in the menstrual cycle.

Perspectives and Significance

A recent report showed a significant relationship between orthostatic tolerance and VDR activity in healthy humans (57). Thus, the VDR is speculated to contribute to BP regulation during orthostasis via venous distension of the lower body capacitance vessels by the gravity-induced volume shift (57). Abnormalities in BP regulation during orthostasis can cause orthostatic hypotension, which is known to be associated with all-cause mortality, coronary artery disease, heart failure, and stroke (58). Thus, the presented data suggest that future studies evaluating baroreflex function during venous distension in patients with cardiovascular (CV) disease could provide new and clinically relevant insights on mechanisms of autonomic dysfunction. On the other hand, impaired CBRS contributed to adverse CV outcomes such as life-threatening arrhythmias and sudden death (59, 60). In addition, impaired SBRS was associated with a variety of CV diseases, including hypertension (61) and heart failure (62), and impaired SBRS was related to heart failure severity (63). Moreover, baroreflex function is regarded as an important prognostic factor (64).

Study Limitation

There are several limitations in the present study. First, possible activation of mechanoreceptors and metaboreceptors by the occlusion cuff itself cannot be excluded. However, in previous studies, the degree of the BP response to metaboreflex activation by postexercise ischemia depended on the intensity of the prior exercise (65), and limb occlusion by a cuff without exercise did not increase BP (66). The earlier reports (2, 6, 7, 25–28) and the present data showed that the occlusion cuff itself did not affect BP and MSNA. Therefore, we speculate that activation of group III and IV afferents by the occlusion cuff itself, does not play a predominant role in altering SBRS and CBRS in this study. Second, both CBRS and SBRS were assessed during spontaneous BP changes. Spontaneous BP changes tend to be small and center around the operating points. Thus, in the present study, baroreflex curves were not assessed across a large range of pressures. In future studies, baroreflex function across a larger pressure range could be assessed with other approaches (e.g., neck pressure/suction). Third, the VDR period was shorter than other periods. In previous reports, the effect of the shorter sampling period contributed to the lower reproducibility of the calculated SBRS (13, 67). We cannot completely exclude the possible effect of the shorter VDR period and the smaller total number of MSNA bursts during the VDR on the BRS results in this study. However, as described under methods, we included only slopes with high R values (SBRS, R ≥ 0.5, CBRS sequences, R² > 0.8). The mean R values for SBRS were all >0.88 during all three measurement periods. The SD of the average slopes was also similar among the three periods (Fig. 4), which suggests that the scattering of the slope values did not differ among periods. We acknowledge that the detected sequence numbers for CBRS during the three periods were different (the average number of CBRS sequences in baseline, Pre-Inf, and VDR was 25, 20, and 6, respectively). However, to our knowledge, there is no evidence from earlier reports that CBRS calculated from a shorter period would be lower than that from a longer period of measurement. Moreover, the SD of average CBRS during the three periods was also similar (Fig. 4). Therefore, we believe that the difference in the lengths of the recording periods did not significantly affect the final results (i.e., the changes in SBRS and CBRS). Finally, we only included healthy young subjects in our study. Whether similar results will be seen in patients with cardiovascular diseases and other diseases is unknown and should be addressed in the future.

Conclusion

In conclusion, the present data show that venous distension via venous saline infusion into a forearm during circulatory occlusion (i.e., the venous distension reflex, VDR) increased the sensitivity of baroreflex control of MSNA and decreased the sensitivity baroreflex control of HR. We speculate that these changes in the baroreflex sensitivities and the resetting of the operating points contribute to the modulation of MSNA and BP during limb venous distension. Further studies are needed to examine the effects of venous distension on baroreflex function in patients with cardiovascular diseases.

GRANTS

This work was supported by National Institutes of Health Grants R01 HL144781 (to J.C. and L.I.S.) and UL1 TR002014 (to L.I.S.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.I.S. and J.C. conceived and designed research; U.A.L., C.B., J.C.L., and J.C. performed experiments; T.H., C.B., J.C.L., and J.C. analyzed data; T.H., U.A.L., J.C.L., L.I.S., and J.C. interpreted results of experiments; T.H. and J.C.L. prepared figures; T.H., U.A.L., J.C.L., L.I.S., and J.C. drafted manuscript; T.H., U.A.L., L.I.S., and J.C. edited and revised manuscript; T.H., U.A.L., C.B., J.C.L., L.I.S., and J.C. approved final version of manuscript.