Data Collection & Analysis in Human Autonomic Research: How to Guide to Successful Testing

Jackie Limberg, Ph.D.
Assistant Professor
Copyright 2020 J. Limberg and InsideScientific. All Rights Reserved.
Data Collection & Analysis in
Human Autonomic Research:
How to Guide to Successful
Testing

Dr. Jackie Limberg discusses the basics
of human autonomic research and
shares tips & tricks for data
collection and analysis during
human autonomic testing to
improve the accuracy and
reliability of your findings.
Copyright 2020 J. Limberg and InsideScientific. All Rights Reserved.
Data Collection & Analysis in
Human Autonomic Research:
How to Guide to Successful
Testing

Learning Objectives
 Select equipment appropriate for the research questions
being addressed
 Create a research setting that is conducive to reliable and
accurate human autonomic data
 Identify tests appropriate for measuring basal versus reflex
autonomic control
 Describe the pros and cons of tests used in human autonomic
research
 Anticipate and avoid areas that may increase data variability
and reduce your ability to interpret results

Acronyms
ECG Electrocardiogram
HR Heart rate
HRV Heart rate variability
BP Blood pressure
MBP Mean blood pressure
NIBP Non-invasive blood pressure
NICO Non-invasive cardiac output
MSNA Muscle sympathetic nerve activity
MVC Maximal voluntary contraction
PEI Post-exercise ischemia
VT Tidal volume
O2 Oxygen
CO2 Carbon dioxide
SpO2 Oxygen saturation

• An important tool the central
nervous system uses to
maintain homeostasis at rest
and in response to changes
in physiological state.
• Homeostasis is achieved
through changes in activity
of the parasympathetic and
sympathetic nervous system
branches.
What is the Autonomic Nervous System?

• Activity: natural or normal
function, such as a process that
an organism carries on or
participates in by virtue of being
alive
• Sensitivity: the capacity of an
organism or sense organ to
respond to stimulation
• Reactivity: the quality or state
of being reactive or readily
responsive to a stimulus,
occurring as a result of stress
Merriam-Webster Medical Dictionary
What is the Autonomic Nervous System?

Barretto et al. International J Cardiology. 2009.
Muscle sympathetic nerve activity
(MSNA) is predictive of mortality.
Activity
Baroreflex sensitivity is predictive
of mortality.
Osterziel et al. Br Heart J. 1995.
Sensitivity
“Variability in autonomic responses to stress may provide a
unique window of insight into hypertension and other
cardiovascular diseases” – Carter & Goldstein (2015)

2
3
4
1
Levick, JR. “Introduction to Cardiovascular
Physiology” Arnold 4th edition.

Activity
Sensitivity
Reactivity

Key Equipment
• Select equipment appropriate for the
research questions being addressed

Limberg Lab
MU-PAW
Gwynn Hall
University of Missouri

PowerLab
16/35
8/35
Limberg Lab
MU-PAW
Gwynn Hall

Limberg Lab
MU-PAW
Gwynn Hall
Stimulus
Isolator
(MSNA)
Grip force
transducer
“Switching
Valve”
(Automated Pneumatic
Controller)
8/35

Limberg Lab
MU-PAW
Gwynn Hall
16/35

Human NIBP
Unit
(Beat-to-beat BP)
ECG Bio Amp
(Heart Rate) NeuroAmp Ex
(MSNA)
PowerLab 16/35
2
3
4
1
3
Limberg Lab
MU-PAW
Gwynn Hall
Primary Components
5
Respiratory Belt
Transducer
(Pod DIN Port)

ECG
BP
Belt
MSNA
Limberg Lab
MU-PAW
Gwynn Hall

Oximeter Pod
(Finger/Ear SpO2)
Pneumotach
(Flow, Volume)
8
7
6
Automated
Pneumatic
Controller
Limberg Lab
MU-PAW
Gwynn Hall
Secondary Components

Oximeter Pod
(Finger/Ear SpO2)
Pneumotach
(Flow, Volume)
8
7
6
Automated
Pneumatic
Controller
Limberg Lab
MU-PAW
Gwynn Hall
Secondary Components
GEMINI
Gas Analyzer
(Inspired/Expired
O2/CO2)
9
Drying Tube
Desicant Cartridge
In-line filters
Dehumidifier

Pneumotach
Non-latex
Gas Reservoirs
Pulse Oximeter
Belt
Automated
Pneumatic
Controller
Inspired/Expired
Gasses
Limberg Lab
MU-PAW
Gwynn Hall

LabChart – Data Collection
ECG
BP
Belt
O2
CO2
VT
MSNAInt
MSNAFiltered
Sample rate: 10 kHz
Digital Filter, Band-pass (0.5 to 2 kHz)
Shift (~1.2 s)
Standard Integral, Time Constant Decay (0.1 s)
Integral, Reset Each Cycle
2-point calibration, Shift (-1 s)
Sample rate: 1 kHz
Band-pass filter (0.5 to 35 Hz)

LabChart – Data Collection
ECG
BP
Belt
O2
CO2
VT
MSNAInt
MSNAFiltered
Sample rate: 10 kHz
Digital Filter, Band-pass (0.5 to 2 kHz)
Shift (~1.2 s)
Standard Integral, Time Constant Decay (0.1 s)
Integral, Reset Each Cycle
2-point calibration, Shift (-1 s)
500:1
10k:1
Cyclic Measures (Rate, Human ECG)
Cyclic Measures (Mean, Arterial Pressure)
REST EXERCISE ISCHEMIA

2 monitors
Limberg Lab
MU-PAW
Gwynn Hall

Getting Started
• Create a research setting that is conducive to reliable
and accurate human autonomic data
• Anticipate and avoid areas that may increase data
variability and reduce your ability to interpret results
Hart EC, et al. Am J Physiol Heart Circ Physiol, 2017.
Limberg JK, et al. Am J Physiol Heart Circ Physiol, 2020.

Laboratory Conditions
• Quiet
• Temperature controlled (21 - 24°C)
• Void of distractions
• Examples: conversations, loud music, people
coming in and out of the laboratory
• All recordings completed during quiet
rest; however, participants should not
fall asleep

Edner DN, et al. Int J Psychophysiol, 2009.
Laboratory Conditions

Quiet
Rest
Talking ECG
BP
Belt
O2
CO2
Vt
ECG
BP
Belt
O2
CO2
Vt
LabChart, ADinstruments.

Outside influences
• Participants should refrain from:
• Alcohol, caffeine, intense exercise, and
smoking/nicotine for at least 12 hours before an
experiment.
• Food/drink for at least 3 hours before an experiment.
• Experiments scheduled for the same time of day, after a
normal night’s sleep, during wakefulness.
• Time-controlled experiments may be necessary to account
for the effect of prolonged sedentary time on outcome
variables.
• All participants should be asked to void their bladder.

Micturation
LabChart, ADinstruments
Baseline ~20 min later
ECG (Heart Rate)
Finger Blood Pressure
Respiratory Belt
Muscle Sympathetic
Nerve Activity
Total Peripheral Resistance
(NICO Software Extension)
~5 min later
Minute 48 Minute 68 Minute 74
Minute 84: “I need to use the restroom”

Selecting participants
• It is standard practice to inquire about past and
current medical history and current medications
to determine whether individuals meet eligibility
criteria.
• Medications known to impact the autonomic,
cardiovascular, and endocrine systems should
be documented (including oral contraceptives,
hormone replacement therapy).

What is your question?
• Identify tests appropriate for measuring basal versus
reflex autonomic control
• Describe the pros and cons of tests used in human
autonomic research
• Select equipment appropriate for the research questions
being addressed

Basal
Activity

Activity: natural or normal function, such as a process that an
organism carries on or participates in by virtue of being alive
• Sympathoadrenal activity (Sympathetic)
• Plasma and/or urine norepinephrine
• Neural recording
• Cardiac vagal activity (Parasympathetic)
• Heart rate variability
Muscarinic receptor antagonists (Atropine)
Plasma norepinephrine spillover (Tritiated/radiolabeled norepinephrine)
Adrenergic receptor antagonists (Propranolol, Phentolamine)

Activity: natural or normal function, such as a process that an
organism carries on or participates in by virtue of being alive
• Sympathoadrenal activity (Sympathetic)
• Plasma and/or urine norepinephrine
• Cardiac vagal activity (Parasympathetic)
• Heart rate variability

Fig 2B: A cardioinhibitory motor axon supplying the sinus
node of the heart (contribute to parasympathetically‐mediated
decreases in heart rate)
Fig 1: Schematic and ultrasound images of the orientation of the head,
location of the vagus nerve, and the location of the microelectrode.
https://www.youtube.com/watch?v=ghGAykjU9ZY&feature=youtu.be
“Physiology Shorts”

Heart Rate Variability (HRV)
• Premise: Beat-to-beat variation in the length of
cardiac cycles.
• Interpretation: The greater the HRV, the greater
the modulatory influence of the cardiac vagus on
heart rate.
• A valuable, non-invasive measure of non-
stationary balance between sympathetic and
parasympathetic branches of the autonomic
nervous system.
Heart rate variability. European Heart Journal (1996)
Heart Rate Variability Today. Progress in Cardiovascular Diseases (2012)
Heart rate variability: origins, methods, and interpretive caveats. Psychophysiology (1997)

https://www.adinstruments.com/support/videos/hrv-20
The HRV 2.0 Add-On for LabChart
May 2014
Heart Rate Variability

 Sample Rate:
 Continuous ECG recording
 Sampled at 500-1000 Hz (1-2 ms resolution)
 Minimum 200 Hz – anything less will create “jitter” in the QRS complex
 Filter:
 Band pass 0.5-35 Hz, High pass 0.5 Hz
 Check the frequency response of the filter, verify spectral components are not altered
 Duration:
 Short-term recording lengths standardized (5 min or 250 cardiac cycles)
 Physically stable conditions (avoid transient stimuli that evoke a HR change)
 Visual checks and manual corrections (remove ectopic beats, arrhythmias, noise)
 Abnormal rhythms will distort R-R intervals and violate assumptions
 Respiration:
 Potential influence of respiration must be considered (talking = misinterpretation)
 Concurrent measurement of respiratory frequency
 When respiration not recorded, breathing rate should be controlled (15 breaths/min)
NOTES:
Indirect (Non-vagal influences)
Physiological artifacts
Dependence on steady-state (fast HR = low HRV)

Consists of:
• Time intervals between successive normal QRS complexes (R-R intervals).
• Statistical operations to measure dispersion of R-R intervals.
Some things to consider:
• Time-domain analysis is typically measured over 24-hours.
• The total variance is directly related to the length of analyzed recording.
• Recording lengths must be standardized.
• Data should be selected from ~5 min or at least 250 cardiac cycles.
Heart Rate Variability: Time Domain

SDRR: standard deviation of sinus beats (R-R intervals)
(measure of overall variability; Abnormal: <100 ms)
RMSSD: root mean square of successive R-R interval
differences (measure of short-term variability; Abnormal:
<25 ms)
pRR50: percent of adjacent R-R intervals that differ by
more than 50 ms (Abnormal: <3%)
Sympathetic/Parasympathetic
Parasympathetic
Heart attack survivors with SDNN values >100 ms had a
5.3 times lower risk of mortality at follow-up than those
with values under 50 ms
Heart Rate Variability: Time Domain

https://www.adinstruments.com/support/videos/hrv-20
The HRV 2.0 Add-On for LabChart

• Preferred method to the time-domain method when investigating short-
term recordings.
• Approach: Heart rate signal is broken into its constituents (frequencies)
and quantified relative intensity (power).
• Influenced by breathing
• Reported in:
• Absolute values of power (ms2)
• Normalized units [nu; Frequency ÷ (total power-VLF)x100].
Heart Rate Variability: Frequency Domain

HF: High frequency (0.15 – 0.50 Hz), correlates with RMSSD
and pRR50, vagal modulation (not vagal tone)
LF: Low frequency (0.04 – 0.15 Hz), correlates with SDRR
LF/HF ratio:
Low = parasympathetic dominance
High = sympathetic dominance
Parasympathetic
Sympathetic/Parasympathetic
Controversy as to validity of LF measurements reflecting
cardiac sympathetic modulation
Heart Rate Variability: Frequency Domain

Plasma Norepinephrine
• Norepinephrine: primary neurotransmitter released from
postganglionic sympathetic neurons
• Accurate estimate of sympathetic nervous system activation
• Very responsive to lifestyle factors
• Need to standardize: smoking, posture, temperature, dietary intake, medications,
emotional stress/distress, ambient temperature, etc
Bernardi L, et al. Diabetes Metab Res Rev. 27: 654-64, 2011.
Doug Seals, PhD. “Chapter 9: The Autonomic Nervous System” ACSM Advanced Exercise Physiology (2006).

• Approximately 50% of plasma norepinephrine from antecubital venous blood
is derived from sympathetic nerves in the arm
• Whole-body plasma norepinephrine responds slowly (minutes) to physiological
maneuvers
• Can be misleading due to differences in release, reuptake, clearance,
metabolism
Plasma Norepinephrine (cont.)

Muscle Sympathetic Nerve Activity (MSNA)
• Microneurography: Direct recordings of the
discharge rates of postganglionic sympathetic neurons
• Premise: Positioning the tip of a microelectrode in a
peripheral nerve (fibular, tibial, median, radial), allows
for direct and continuous measurement of sympathetic
nervous system activity

Muscle Sympathetic Nerve Activity (MSNA)
• Requires training, time, and patience to obtain
consistent and interpretable recordings
• Invasive procedure, cannot be repeated often in
the same person (e.g., 4 weeks)
• Difficultly maintaining recordings, even from
inactive limbs. Must remain relaxed - excessive
movement can dislodge the electrode.

https://www.youtube.com/watch?v=f9mzmptuXbg
Microneurography: Recording Nerve Traffic Via Intraneural Microelectrodes in Awake Human Subjects
Professor Vaughan Macefield (June 2017)
https://www.youtube.com/watch?v=xih5IZpZgqc
Microneurography – LabChart Software Demonstration
Professor Vaughan Macefield (June 2017)
https://vimeo.com/48459797
LabChart Mastery – Neuroscience Fundamentals Using Scope, Peak Analysis, and Spike Histogram
Brandon Bucher (Jan 2012)
https://www.youtube.com/watch?v=0w3oH4wOjdA
LabChart Mastery – Neurophysiology Analysis: Scope View, Peak Analysis, and Spike Histogram
Brandon Bucher (Nov 2013)
Muscle Sympathetic Nerve Activity - Tutorials

Activity
*Plasma catecholamines
NOTES
 Resting
 Awake
 10-15 minutes
Absolute Integral, Time Constant Decay (0.1 s)
Sample rate: 10,000 Hz
Band-pass filtered (0.5 to 2 kHz)
Metronome (15 breaths/min)
Sample rate: 1,000 Hz
High pass filter: 0.5 Hz
ECG
BP
Belt
MSNA
MSNA
Calibrated to upper arm cuff (2-point calibration)
*NICO Extension: Stroke volume, cardiac output, total peripheral resistance
*HRV 2.0: Time and Frequency domain analyses

Neurovascular responsiveness
• The physiological effects of
the autonomic nervous
system depend on:
• The change in the activities of
the autonomic nerves and
their release of
neurotransmitters
• The responsiveness of the
peripheral tissues to this
neurochemical stimulus
Sympathetic Nerve Terminal
Sympathetic “burst”
Neurotransmitter Release
Adrenergic Receptor
Blood Vessel

Transduction: the action or process of
converting a message into another form
Briant LJB et al, J Physiol (2016) Vianna LC et al, Am J Physiol Heart
Circ Physiol (2012)
Fu Q et al, J Physiol (2012)

Sensitivity

Sensitivity: the capacity of an organism
or sense organ to respond to stimulation
• Baroreflex sensitivity
• Spontaneous
• Modified Oxford
• Head-up tilt
• Lower body negative pressure
• Neck suction/pressure
• Valsalva
• Chemoreflex sensitivity
• Apnea
• Hypoxic ventilatory response
• Hypercapnic ventilatory response
• DeJour’s test (acute hyperoxia)

Bernardi L, et al. Diabetes Metab Res Rev, 2011.
Baroreflex Sensitivity
• Premise: Continuous changes in blood pressure elicit reflex changes in
vagal and sympathetic outflow.
• An increase in blood pressure reduces the firing of sympathetic vascular and
cardiac efferents, and increases firing of vagal cardiac efferents.
• Requirements: beat-to-beat heart rate, blood pressure, and sympathetic
activity (MSNA) monitoring.
• To define baroreflex function, one must consider both vagal efferent
(changes in heart rate) and sympathetic efferent (MSNA) activity.

Adapted from: Halliwill JR & Minson CT, J Appl Physiol (2002).
Cardiac-vagal:
Changes in HR in response
to changes in systolic BP
Sympathetic:
Changes in MSNA in response
to changes in diastolic BP
Halliwill JR. J Appl Physiol, 2000.

• Spontaneous approach:
• Recordings during spontaneous breathing for ~10 minutes under monitored
respiration (or controlled breathing at 15 breaths/min)
• Modified Oxford:
• Arterial pressure changes induced by bolus intravenous nitroprusside (100
µg) followed 1-min later by phenylephrine (150 µg)
• Allow assessment across a range of physiologically relevant blood
pressures
https://www.adinstruments.com/support/videos/labchart-mastery-
automating-analysis-baroreflex-sensitivity
LabChart Mastery – Automating Analysis for Baroreflex Sensitivity
Brandon Bucher

Heart
Rate
(beat/min)
100-
80-
60-
110-
-
90-
-
MBP
(mmHg)
Integrated
MSNA
Filtered
MSNA
0s 20
Nitroprusside
Bolus
Phenylephrine
Bolus
Modified Oxford Test
Limberg JK, et al. J Neurophysiol (2019).

Incognito AV et al. Clin Auton Res, 2018.

Sensitivity: the capacity of an organism or sense organ to
respond to stimulation
• Baroreflex sensitivity
• Spontaneous
• Modified Oxford
• Head-up tilt
• Lower body negative pressure
• Neck suction/pressure
• Valsalva
• Chemoreflex sensitivity
• Apnea
• Hypoxic ventilatory response
• Hypercapnic ventilatory response test
• Modified DeJour’s test

Chemoreflex Sensitivity
Ponikowski et al. Circulation. 2001.
Chemosensitivity is predictive of
mortality.
• Premise: Changes in the partial pressures of
oxygen in the arterial blood elicit reflex changes
in vagal and sympathetic outflow.
• A decrease in the partial pressure of oxygen
elicits both hyperventilation and sympathetic
activation.
• Requires continuous monitoring of heart rate,
blood pressure, sympathetic activity (MSNA),
and ventilation.

Voluntary Apnea
Badrov MB, et al. Am J Physiol Heart Circ Physiol (2016).
• Premise: Robust stimulus for efferent sympathetic outflow
• End-inspiratory (EI): unloading baroreceptors, chemoreflex stimulation, increased
central drive-to-breathe
• End-expiratory (EE): chemoreflex stimulation, lack of ventilatory restraint on
sympathetic outflow, increased central drive-to-breathe

Reactivity

Reactivity: the quality or state of being reactive or readily
responsive to a stimulus, occurring as a result of stress
• Isometric handgrip
• Post-exercise ischemia
• Mental stress
• Mental arithmetic
• Stroop color-word conflict test
• Cold pressor test
REST STRESS WASH-OUT PERIOD
2-5 min 10-20 min
2-3 min
5 min each
2-3 min
2 min or to fatigue

• Mental stress
Grotle AK et al, Autonomic Neuroscience, 2020.
Goal: Identify and restore muscle blood flow
and the intramuscular concentration of
metabolites to some optimal level by raising
blood pressure and cardiac output

Isometric Handgrip and Post-Exercise Ischemia (PEI)
 Mechanisms:
 Isometric handgrip: Central command + mechanoreceptor + metaboreceptor activation
 Post-exercise ischemia (PEI): Isolate effects of the metaboreceptors
 Cuff inflated suprasystolic pressure (trap metabolites, maintain receptor activation)
 Considerations:
 Reach exercise target quickly without overshooting
 Maintain constant force (visual feedback)
 Ratings of perceived exertion (BORG scale)
 Reproducibility:
 BP response to exercise/PEI is reproducible within and between visits
 MSNA response to exercise is reproducible between (but not within) visits
 MSNA response to PEI has poor-to-moderate reproducibility
 Static handgrip at 30% MVC may be below the metaboreflex threshold
Dillon GA, et al. J Appl Physiol, 2020.

NOTES:
 Perceived exertion scale
 Exercise duration?
 Exercise workload (≥30%)
 Visual feedback
 Repeat trials?
Grip force
transducer
Cuff
inflation
system

• Mental stress

Cold Pressor Test
NOTES:
 Pain Perception
 Thermal Perception
 Water temperature (0-4○C)
• Premise: Assess sympathetic reactivity to a generalized
sympathoexcitatory stimulus. Document afferent/efferent sympathetic
reflex pathways are intact.
• Mechanism: Activates sensory afferent nerve fibers (cold nociceptors)
• Procedure:
• Immersion of hand or foot passively into a bucket containing an ice slurry for 2 min
• Temperature of the ice bath measured (0-4○C) and amount of ice added
consistently
• Ratings of pain and thermal perception obtained at the end
Reproducibility of BP and MSNA responsiveness is very good
within and between experimental visits.
Dillon GA, et al. J Appl Physiol, 2020.
Participants should avoid isometric contraction, breath-holding
and/or the Valsalva maneuver during the test.
Hand between two gel-packs confined inside an insulated mitt pre-cooled to −12 °C (Fu et al, J Physiol, 2002)

Baseline Cold Pressor Test
ECG
BP
Belt
MSNAFiltered
MSNAInt
NOTES:
 Pain Perception
 Thermal Perception
 Water temperature (0-4○C)
Cold Pressor Test

Mental Stress
 Premise:
 Acute laboratory-based psychological stress with minimal physical movement
 Use of extreme stress has ethical implications in human research
 Reproducibility:
 MSNA response is variable between participants (“responders” and “non-responders”)
 MSNA reactivity to mental stress is consistent within and between visits
 Must assess perceived difficult of the task
Fonkoue I & Carter J. Am J Physiol Regul Integr Comp Physiol, 2015.
Carter & Goldstein. Compr Physiol, 2015.
NOTES:
 Psychological distress survey
 Examples:
 Mental arithmetic tasks: Associated with greater
anxiety levels and BP elevations
 Stroop color-word conflict test

Adapted from: El Sayed K et al, J Physiol (2016).
Mental Arithmetic
Positive MSNA Responder Negative MSNA Responder
A more rapid rise in BP at onset of stress may
occur with baroreflex resetting, leading to
baroreflex suppression of MSNA
Lag in rise in BP may allow time for
baroreflex resetting to occur and with a
higher set point, MSNA may increase.

Summary
 Select equipment appropriate for the research questions being
addressed
 Anticipate and avoid areas that may increase data variability and
reduce your ability to interpret results
 Create a research setting that is conducive to reliable and accurate
human autonomic data
 Identify tests appropriate for measuring basal versus reflex autonomic
control
 Describe the pros and cons of tests used in human autonomic research

Questions?
LimbergJ@Missouri.edu

Thank you for participating!
Before you go…
1. To watch the webinar on
demand, go to:
www. insidescientific.com
2. Interested in learning more?
Visit:
www.adinstruments.com

Data Collection & Analysis in Human Autonomic Research: How to Guide to Successful Testing

Recommended

Recommended

More Related Content

What's hot

What's hot (14)

Similar to Data Collection & Analysis in Human Autonomic Research: How to Guide to Successful Testing

Similar to Data Collection & Analysis in Human Autonomic Research: How to Guide to Successful Testing (20)

More from InsideScientific

More from InsideScientific (20)

Recently uploaded

Recently uploaded (20)

Data Collection & Analysis in Human Autonomic Research: How to Guide to Successful Testing

Editor's Notes