Electrocardiogram signals were continuously recorded at 500 Hz sampling frequency from each rat throughout the course of the study starting from implantation of the wireless ECG device at W2. Telemetric ECG data from 16 rats were recorded simultaneously using IoX software (EMKA Technologies Inc.; France), stored in a PC, and later formatted for further analysis by custom MATLAB scripts. Two weeks after recovery from the second surgery, VNS protocol was initiated at W7 and continued for 8 weeks (Figure 1A ). A wireless pulse generator was connected to each rat by the experimenter and programmed using the software for applying VNS in awake behaving rats. VNS was delivered five times a week (on weekdays except holidays) in the morning (around 10 AM) based on fixed parameters: constant-current, biphasic, and charge-balanced square-wave pulses (1 mA/phase, 100 μs/phase) at 30 Hz applied for 2 min ON/2 min OFF for 10 min (Figure 1C ). To provide Sham animals with similar environmental stimuli, cage lids were removed and the same amount of animal handling (except attaching stimulator) was performed by experimenters. After attaching the wireless pulse generator to the connectors (Figure 1C ), VNS was scheduled by the experimenter and Treatment rats received VNS simultaneously while moving freely inside the cage.
Iox software
Manufactured by EMKA Technologies
Sourced in France
IOX software is a data acquisition and analysis tool developed by EMKA Technologies. It is designed to interface with various types of laboratory equipment and sensors, enabling users to capture, store, and analyze experimental data.
Automatically generated - may contain errors
11 protocols using iox software
1
Vagus Nerve Stimulation in Rats
2
Respiratory Measurements in Mice
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Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, Paris, France) supplied with room air as described previously (Hill et al., 2016 (link), 2018 (link)). Mice were habituated to respiratory chambers for 30 min, on the day prior to experimentation. Respiratory parameters were recorded (IOX software ‐ EMKA Technologies, Paris, France) and averaged over 5 min periods.
Data are presented both as minute volume (MV) and as percentage change from the pre‐drug MV baseline, calculated from data for each individual mouse before collating and plotting as a mean. Mice within cohorts may vary in size and this varies their individual MVs, accordingly; data are presented as a percentage change from each mouse's pre‐drug baseline controls for these inherent variations.
Data are presented both as minute volume (MV) and as percentage change from the pre‐drug MV baseline, calculated from data for each individual mouse before collating and plotting as a mean. Mice within cohorts may vary in size and this varies their individual MVs, accordingly; data are presented as a percentage change from each mouse's pre‐drug baseline controls for these inherent variations.
3
Murine ECG Recording and QT Interval Analysis
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All electrocardiogram (ECG) recording sessions were performed during daytime. Six-lead restrained ECG monitoring of conscious mice was performed with the ecgTUNNEL system platform (EMKA Technologies, France). The animals were allowed to stay in the restraining system for 5 min before starting ECG recordings to minimize the effects of stress. Six leads of ECG were recorded for 20 min using the IOX software (EMKA Technologies). Lead II recordings were analyzed using the Ecg-autosoftware (EMKA Technologies). QT intervals were measured as the time interval between the initiation of the QRS complex and the end of the T-wave, and corrected QT intervals (QTc) were calculated using the formula: QTc = QT/(RR/100)1/2.
4
Respiratory parameters in young mice
5
Arterial Pressure Monitoring in Anesthetized Rats
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Animals were anesthetized with 2% volume of isoflurane and 0.6 L·min−1 O2 to limit hemodynamic effects of anesthesia. Arterial blood pressure measurements were performed through the left carotid artery to calculate the mean arterial blood pressure (MAP) and heart rate (HR). Briefly, the right carotid artery was isolated and ligated at the distal end, and a PE-50 catheter containing Ringer Lactate was inserted. Pressure signal and HR were continuously recorded, displayed, and stored by the IOX® software (EMKA Technologies, Paris, France).
6
Whole-body plethysmography for evaluating mouse respiratory function
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Mouse respiratory function was evaluated by whole-body plethysmography using an EMKA Technologies plethysmograph as described by TREAT-NMD (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.2.002.pdf ). Briefly, unrestrained conscious mice were placed in calibrated chambers containing a pneumatograph that measured pressure differentials within the compartment by a difference in air flow. Mice were allowed to acclimate in chambers for 45 min at a stable temperature and humidity. Data were then collected every 5 s using iox software (EMKA technologies). The inspiration time, Ti, was defined as the start of inspiration to the end of inspiration, and the expiration time was defined as the start of expiration to the end of expiration. The relaxation time, RT, was defined as the time from the start of expiration to the time when 65% of the total expiratory pressure occurred. Pause and enhanced pause (Penh) were defined and calculated by the formulas pause = (Te − RT)/RT and Penh = (PEP/PIP) × pause, where Te is expiratory time, PEP is peak expiratory pressure, and PIP is peak inspiratory pressure. The value of each parameter was calculated from an average of 60 recordings of 5 s representing a total of 5 min. Inclusion criteria for each recording were more than eight respiration events by 5 s and >0% of success rate as measured by iox software.
7
Hypercapnic Ventilation Response in Mice
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Ventilatory responses were measured in conscious mice by whole body plethysmography in chambers manufactured by Data Sciences International (601-1540-001) and recorded with IOX software (EMKA Technologies). A mass flow regulator provided quiet, constant and smooth flow through the animal chamber (0.5 L/min). Mice were familiarized with the plethysmography chamber the day prior to testing (3–4 h acclimation period) and again immediately before the testing protocol (for at least 2 h). The typical protocol entailed three sequential incrementing CO2 challenges (7 min exposures to 4%, 6%, 8% CO2, balance O2; each separated by 5 min of 100% O2). Hypercapnic exposure was performed in hyperoxia to minimize contributions of peripheral chemoreceptors to the hypercapnic ventilatory reflex and attribute ventilatory effects to central chemoreception. CO2 tension in the chambers was verified with a capnograph. After data collection, Poincare analysis of the breathing frequency over the final 3 min of each challenge period for CO2 was performed to select periods of regular, calm breathing for analysis of frequency, tidal volume, and minute ventilation.
8
Murine ECG Monitoring Protocol
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S.c. 4-lead electrocardiograms of isoflurane-anesthetized mice were performed using Emka ECG and recorded using IOX software (Emka Technologies). PR, RR, and QT intervals were measured manually using Ponemah 3 software (Data Sciences International).
9
Cardiac Hemodynamics Assessment in Mice
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Fed mice were anesthetized with 2% isoflurane in 100% O2 at a flow rate of 2 L/min at 14 weeks of age. Body temperature was monitored and maintained at 37 °C using a heating pad. Cardiac hemodynamics parameters were measured using a microtip pressure transducer catheter (1.4-F; Millar Instruments) inserted into the left ventricle through the carotid artery as previously described84 (link). Data were collected and analyzed using IOX software (v.2.10.8.25; emka TECHNOLOGIES, Falls Church, VA). Blood pressure, intraventricular pressures (systole, diastole), and heart rate (HR) measurements were taken when a steady and average value was reached. Subsequent mice were euthanized, and organs were harvested as described in the animal model section.
10
Plethysmography in Respiratory Mice
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Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, Paris, France) supplied with room air or a 5% CO2 in air mixture (Figure S5 ) as described previously (Hill et al., 2016 (link)). Mice were habituated to respiratory chambers for 30 min the day prior to experimentation. Respiratory parameters were recorded (IOX software—EMKA Technologies, Paris, France) and averaged over 5‐min periods. On the day of the experiment, respiration was measured for 20 min prior to drug administration, of which the last 10 min were taken as the baseline.
Data are presented as percentage change from the pre‐drug minute volume baseline, calculated from data for each individual mouse before data being collated and plotted as a mean. Data are presented as a percentage change from each mouse's pre‐drug baseline controls to account for variations in individual minute volume due to variations in mouse size. Raw minute volume data are presented in FigureS1 .
Data are presented as percentage change from the pre‐drug minute volume baseline, calculated from data for each individual mouse before data being collated and plotted as a mean. Data are presented as a percentage change from each mouse's pre‐drug baseline controls to account for variations in individual minute volume due to variations in mouse size. Raw minute volume data are presented in Figure
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