Carotid-body modulation through meditation in stage-I hypertensive subjects: Study protocol of a randomized and controlled study

Adjunctive therapy for hypertension is in high demand for clinical research. Therefore, several meta-analyses have provided sufficient evidence for meditation as an adjunct therapy, without being anchored on reliable physiological grounds. Meditation modulates the autonomic nervous system. Herein, we propose a hierarchical-dependent effect for the carotid body (CB) in attenuating blood pressure (BP) and ventilatory variability (VV) fine-tuning due to known nerve connections between the CB, prefrontal brain, hypothalamus, and solitary tract nucleus. The aim of this exploratory study was to investigate the role of CB in the possible decrease in BP and changes in VV that could occur in response to meditation. This was a prospective, single-center, parallel-group, randomized, controlled clinical trial with concealed allocation. Eligible adult subjects of both sexes with stage 1 hypertension will be randomized into 1 of 2 groups: transcendental meditation or a control group. Subjects will be invited to 3 visits after randomization and 2 additional visits after completing 8 weeks of meditation or waiting-list control. Thus, subjects will undergo BP measurements in normoxia and hyperoxia, VV measurements using the Poincaré method at rest and during exercise, and CB activity measurement in the laboratory. The primary outcome of this study was the detection of changes in BP and CB activity after 8 weeks. Our secondary outcome was the detection of changes in the VV at rest and during exercise. We predict that interactions between hyperoxic deactivation of CB and meditation; Will reduce BP beyond stand-alone intervention or alternatively; Meditation will significantly attenuate the effects of hyperoxia as a stand-alone intervention. In addition, VV can be changed, partially mediated by a reduction in CB activity. Trial registration number: ReBEC registry (RBR-55n74zm). Stage: pre-results.

Twelve normotensive/prehypertensive subjects underwent an RVV reproducibibility study second the following protocol, performed twice, one-week apart, under the same conditions of the laboratory. The subjects were admitted at a climatized lab room at ~7:00 hs after a light breakfast without stimulant foods, and were instructed to remain calm during the study protocol. They were monitored through a QUARK metabolic system (QUARK CPET, COSMED, Rome, Italy, 2016) for breath-by-breath oxygen consumption (V´O2), minute-ventilation (V´E), tidal volume (VT), breathing frequency (fR), end-tidal carbon dioxide (PETCO2) and inspiratory/expiratory time (s), using a low-resistance calibrated turbine (COSMED, Rome, Italy, 2016). The analyser was calibrated with 2-point precision gases (GAMA GASES, São Paulo, Brasil). The turbine was attached to a non-rebreathing two-way valve connected to a naso-oral mask attached to the head with head straps (Hans-Rudolph inc., USA, 2036 series, 2019). All the trials were registered in real-time and saved for posterior analysis, in accordance with previous published methods [S1] .
Resting variability will be measured based on our previously described method [S2] .
First, non-linear evaluation of breath-by-breath signal using an in house Poincaré analysis algorithm will be performed using the "R ® " free software program (http://www.R-project.org/) to calculate the SD1 for minute-ventilation. The resting trial was performed at a seated position for 5 min.
As results, SD1 for minute-ventilation (L/min) showed an average difference between Test 1 and Test 2 of 0.08±0.9 L, and limits of agreement (LOA) of -1.8/1.7 L (See Supplementary Figure 1).
S. Figure 1 Bland-Altman plot for Poincaré variability analysis in a test-retest design for minute-ventilation SD1. T1 = data collection at Time 1 and T2=data collection at Time 2.

2.Reproducibility for blood pressure without and with paced breathing.
Blood pressure were measured based under the same described metabolic and ventilatory RVV monitoring. The arm was positioned at the heart level and BP measures (3x, 1-min apart) only under normoxic conditions were measured, immediately after the 5-min RVV acquisition. The target was a minimum of three BP measures differing within < 5 mmHg and none BP measure differing within > 10 mmHg [S3, S4] between three, 1-min apart, successive oscillometric measurements, (Multiparametric Monitor, Dixtal-Philips ® , Manaus, Brazil, 2015). Aditional measurements were performed only if the first two reading differ by > 10 mmHg and BP was recorded as the average of the last two BP readings [S3] . After 10-min resting, the subjects underwent the same protocol for blood pressure measurements under paced breathing frequency (20 breaths min -1 ), with controlled inspiratory time (duty cycle~0.3) throughout metronome pacing with incentive screens (Bounce Metronome, USA, 2015). The lights were dimmed to ensure stable measurements, and no interaction with the participants will occur during the assessment.
As results, blood pressure without paced breathing showed an average difference between Test 1 and Test 2 of -0.58±4.5 mmHg, and limits of agreement (LOA) of -9.5 /8.5 mmHg (See Supplementary Figure 2). For blood pressure under paced breathing, the average difference between Test 1 and Test 2 was -0.50±4.2 mmHg, and limits of agreement (LOA) of -8.6/7.6 mmHg (See Supplementary Figure   3). The blood pressure during paced breathing was measured under similar average minute-ventilation in comparing Test 1 (14.8±5.4 L) and Test 2 (15.0±6.3 L) trials (p-value=0.867).
S. Figure 2 Bland-Altman plot for reproducibility analysis without paced breathing, in a test-retest design for systolic blood pressure (SBP). Data collection at Time 1 and Time 2.