Surgical preparation

Animal care was provided in strict accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, which has been approved by the Physiological Society of Japan. The experimental protocols were reviewed and approved by the Animal Subject Committee of the National Cerebral and Cardiovascular Center (No. 20008, 21009).

Eight male Wistar-Kyoto rats (304–395 g) were anesthetized with an intraperitoneal injection (2 mL/kg) of a mixture of urethane (250 mg/mL) and α-chloralose (40 mg/mL). The anesthetic mixture was diluted 18-fold and administered continuously (2 mL·kg−1 h−1) from the right femoral vein. The rats were mechanically ventilated with oxygen-supplied air. AP was measured from the right femoral artery, and the heart rate (HR) was detected from a body surface electrocardiogram. A pair of stainless-steel wire electrodes (AS633, Cooner Wire, CA, USA) was attached to a postganglionic branch of the left splanchnic sympathetic nerve and fixed with silicone glue (Kwik-Sil, World Precision Instruments, FL, USA). The nerve activity was amplified using a biosignal amplifier (AB-610J, Nihon Kohden, Japan) with a bandpass filter setting between 150 and 1000 Hz. The amplified signal was then full-wave rectified and low-pass filtered at a cut-off frequency of 30 Hz to quantify SNA. Bilateral carotid sinus baroreceptor regions were isolated from the systemic circulation [9, 10], and the carotid sinus pressure (CSP) was controlled with a servo-pump system (ET-126, Labworks, Costa Mesa, CA, USA). Bilateral vagal and aortic depressor nerves were sectioned at the neck to minimize reflex effects from the cardiopulmonary region and aortic arch.

Protocol

After completing the surgical preparation, we waited for at least 30 min to obtain stable hemodynamics. During the waiting period, CSP was servo-controlled to mimic instantaneous AP. Although pressure waveforms did not exactly match between the CSP and AP signals due to the limited performance of the servo controller, the baroreflex negative feedback loop was effectively closed.

To estimate the open-loop static characteristics of the carotid sinus baroreflex, CSP was first decreased to 60 mmHg for 5 min, and then increased stepwise to 180 mmHg in increments of 20 mmHg every minute. The stepwise CSP input was repeated, and the sequences were referred to as S1 to S6 (Fig. 1, left panels). One minute after the completion of S2, low-dose (2 μg/kg) clonidine (clonidine hydrochloride, Tokyo Chemical Industry; 2 μg/mL dissolved in physiological saline) was administered from the left femoral vein. One minute after the completion of S4, high-dose (5 μg/kg) clonidine (5 μg/mL dissolved in physiological saline) was added. The effect of high-dose clonidine may need to be interpreted as a cumulative one.

Fig. 1
figure1

Time series of carotid sinus pressure (CSP), sympathetic nerve activity (SNA), arterial pressure (AP), and heart rate (HR) obtained in one rat. In the left panels, stepwise CSP changes were repeated and referred to as S1 to S6. One minute after the completion of S2, low-dose (2 μg/kg) clonidine was intravenously administered. One minute after the completion of S4, high-dose (5 μg/kg) clonidine was intravenously administered. The baroreflex response in S2 was treated as the control, whereas the responses in S4 and S6 were used to evaluate the effects of low-dose and high-dose clonidine, respectively. In the right panels, hexamethonium bromide (C6) was intravenously administered. CSP was servo-controlled to mimic instantaneous AP during the C6 protocol. The CSP and AP signals are virtually the same at this time resolution. SNA was depicted as a 10-Hz resampled signal (gray) and a 2-s moving averaged signal (black). AP and HR were depicted as 200-Hz resampled signals (gray) and 2-s moving averaged signals (black)

At the end of the protocol, CSP was again servo-controlled to mimic instantaneous AP. Under the baroreflex closed-loop condition, hexamethonium bromide, a ganglionic blocker, was intravenously administered (60 mg/kg), and the noise level of SNA was assessed. Approximately 3 min after the injection of hexamethonium bromide, mean AP was obtained as a 10-s averaged value.

Data analysis

Data were recorded on a laboratory computer system using a 16-bit analog-to-digital converter. We treated the baroreflex response in S2 as a control and evaluated the effects of low-dose and high-dose clonidine from the responses in S4 and S6, respectively. In each of S2, S4, and S6, the SNA, AP, and HR data were averaged for the last 10 s at each CSP level. As the absolute amplitude of SNA varied among animals depending on the recording conditions, SNA was normalized in each animal based on the value at the CSP of 60 mmHg in S2 (100%) and the value after ganglionic blockade (0%). The relationships between CSP and AP (the total reflex arc), between CSP and HR, and between CSP and SNA (the neural arc) were analyzed using a four-parameter logistic function as follows [11, 12]:

$$y = frac{{P_{1} }}{{1 + exp left[ {P_{2} left( {{text{CSP}} – P_{3} } right)} right]}} + P_{4} ,$$

where y denotes the output variable, and P1, P2, P3, and P4 represent the response range, slope coefficient, midpoint pressure on the CSP axis, and the lower asymptote of the sigmoid curve, respectively.

The relationship between SNA and AP (the peripheral arc) was quantified using linear regression analysis [12]:

$${text{AP}} = b_{0} + b_{1} times {text{SNA,}}$$

where b0 and b1 represent the intercept and slope of the regression line, respectively.

The operating point of the carotid sinus baroreflex was determined from the intersection point between the fitted neural and peripheral arcs on a baroreflex equilibrium diagram [12,13,14]. The operating-point AP values were estimated under conditions of control, after low-dose clonidine, and after high-dose clonidine. To exclude the peripheral effect of clonidine, the intersection points after clonidine were also estimated by reflecting changes in the neural arc alone.

Statistical analysis

Data are presented as box and whisker plots except otherwise specified. The fitted parameter values of the baroreflex static characteristics were compared among conditions of control, after low-dose clonidine, and after high-dose clonidine using one-way repeated-measures analysis of variance (ANOVA) followed by a Tukey test. The Geisser-Greenhouse’s correction was applied (Prism 8, GraphPad Software, San Diego, CA, USA). The operating-point AP values estimated under conditions of control, after low-dose clonidine (with or without the peripheral effect), and after high-dose clonidine (with or without the peripheral effect) were compared using one-way repeated-measures ANOVA followed by a Tukey test. The AP value obtained after the injection of hexamethonium bromide was compared with the intercepts of the peripheral arcs determined under the control condition and after high-dose clonidine using one-way repeated-measures ANOVA followed by a Tukey test. The differences were considered statistically significant at P < 0.05.

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