research thesis proposal written and oral.
Breathing Patterns to Minimize Cardiovascular Disease Risk in Major Depressive Disorder
Methods
The proposed study will evaluate the effects of 15 minutes of device-guided slow breathing and 15 minutes of mindful
meditation (MM) (independent variables) on respiratory rate (breaths/min), MSNA burst frequency (bursts/min) and
incidence (bursts/100 heart beats), blood pressure (mmHg), and heart rate (beats/min) (dependent variables) during the
cold pressor test (CPT). The primary outcome of this project is MSNA burst frequency (bursts/min).
A total of 25 young adults (18-35 years old) with depressive symptoms will be recruited for this study. Participants will be
recruited from the University of Texas at Arlington and the surrounding area through the use of flyers, email, social
media, class presentations, and radio ads. Inclusion criteria consists of those 18-35 years old and confirmation of
depressive symptoms with a diagnostic interview. Exclusion criteria consists of a mental illness aside from depression,
any use of depression mediation, active suicidal/homicidal intent, active alcohol or drug dependence, an eating disorder,
use of medication that could alter how the brain or cardiovascular system functions, cardiovascular, kidney, lung or
metabolic disease, tobacco use, pregnancy, or amenorrhea (women).
Sample size was calculated a priori using G-power. MSNA measurement in young adults with MDD has not been studied
to our knowledge; therefore, due to the similarities between posttraumatic stress disorder (PTSD) and MDD, MSNA
values were estimated from studies involving young adults with PTSD (Fonkoue, Marvar et al., 2018). Estimates of MSNA
values during the DGSB+CPT and MM+CPT conditions were derived from both previous studies of MSNA recordings
during both breathing patterns, and clinically significant MSNA values (Park et al., 2014; Fonkoue, Marvar et al., 2018).
Means were estimated as the change score between a cold-pressor test and the DGSB+CPT condition, and the change
score between a cold-pressor test and the MM+CPT condition. Accounting for a 70% success rate on peroneal nerve
innervation and MSNA recording, a total of 25 subjects are needed to detect a 3 burst/min difference between DGSP
and MM conditions with 80% power and an alpha level of 0.05.
Experimental Approach
Participants will arrive to the laboratory for one screening visit and two experimental visits. Each participant will be
randomly allocated to the device-guided slow breathing or mindful meditation intervention for Experimental Day 1 using
a random number generator. Each participant will participate in the opposite intervention for Experimental Day 2, and
each experimental visit will be separated by at least 24 hours. Participants will be instructed to avoid exercise and
alcohol consumption for 24 hours prior to the experimental visits, and abstain from food and caffeine 12 hours prior to
the experimental visits.
The screening visit will consist of basic health assessments and take about one hour. First, height weight, 7-site skinfold
(body fat percent), blood pressure, heart rate and temperature will be taken. Then, the participant will complete a
medical health history form to confirm the absence of any exclusion criteria. Women will also complete a urine
pregnancy test to confirm absence of pregnancy. Last, the MINI international neuropsychiatric interview will be
administered by a trained research member to determine the presence of depressive symptoms and absence of other
mental illnesses. Lastly, a blood draw will be taken for assessment of a complete metabolic panel, lipid profile, and
hemoglobin A1c.
Both experimental visits are identical with only the intervention (device-guided slow breathing vs mindful meditation)
during the cold pressor test varying. All participants will arrive to the laboratory in the morning having avoided exercise,
alcohol, food, and caffeine as outlined above. After using the restroom, participants will lay supine and be instrumented
with a tungsten microneurography electrode in the peroneal nerve for MSNA, the Finapres for beat-to-beat blood
pressure, electrocardiograph electrodes for continuous heart rate and sinoatrial rhythms, and a respiratory belt to
measure respiration rate. After instrumentation, 15 minutes of baseline MSNA, BP, HR, and respiration data will be
collected. Following baseline, a “baseline” cold pressor test will be performed for 2 minutes to obtain baseline
cardiovascular reactivity. Next, there will be 15 minutes of rest to ensure MSNA, BP, and HR return to resting levels.
Lastly, participants will perform 15 minutes of device-guided slow breathing or guided mindful meditation. During the
last 2 minutes of device guided slow breathing and mindful meditation, the participant will perform another CPT.
Participants will be asked to measure their perceived stress on a scale of 1-10, with 1 being “not stressed at all”, to 10
being the “most stressed they’ve ever been”, after each CPT. MSNA, blood pressure, heart rate, respiratory rate, and
ECG will be collected for the duration of the visit. A study schematic of the experimental visit can be found below in
Figure 1.
Figure 1. Schematic of the experimental visit. Abbreviations: electrocardiogram (ECG), blood pressure (BP), muscle sympathetic nerve activity
(MSNA), heart rate (HR), cold pressor test (CPT), slow breathing (SB).
7-site skinfold is a validated measure of total body fat percentage. The method consists of gathering subcutaneous fat
into a “pinch” and measuring the thickness (mm) of the pinch with calipers across 7 standardized sites of the body
(chest, triceps, subscapular, superiliac, thigh, midaxillary, and abdominal). Each site is measured 2-3 times in a rotating
fashion. The measures are then inserted into an algorithm established by the American College of Sports Medicine to
produce a body fat percentage. Since the reliability of this method is dependent upon the individual performing the
measure, one trained research personnel will complete the body composition portion of the experiment for all subjects.
The MINI international neuropsychiatric interview is a short, structured interview (~15 minutes) with 16 modules
assessing DSM-IV and ICD-10 psychiatric disorders. It has been validated in many clinical populations including those
with MDD (Fantino & Moore, 2009; Sheehan et al., 1998). The MINI will be administered by a research team member
who has received extensive training in the administration of this interview by a psychiatrist. The results from the MINI
will be used to document MDD severity and determine depressive symptoms for the purpose of this study.
A small amount of blood (~10 mL or ~2 teaspoons) will be drawn by a certified phlebotomist at the end of the screening
visit. The blood specimens will then be sent to LabCorp to assess a complete metabolic panel (glucose, BUN, Chloride,
Calcium, Albumin, Bilirubin, AST, Potassium, Alkaline Phosphatase, Sodium, Protein, Creatinine, BUN/Creatinine Ratio,
Globulin, A/G ratio), lipid panel (total cholesterol, triglycerides, HDL cholesterol, Cholesterol/HDL ratio, LDL cholesterol,
VLDL cholesterol), and hemoglobin A1c. All blood test results will be provided to the participant.
Microneurography allows for the direct recording of efferent sympathetic nervous system activity in humans. Multiunit
postganglionic MSNA will be recorded using standard techniques described within previous literature (Sundlof & Wallin,
1978; Vallbo et al., 1979). A wand-like device is used for external electrical stimulation to track the anatomy of the
peroneal nerve. An active tungsten microelectrode and grounding reference electrode are inserted through the skin into
the peroneal nerve and surround tissue, respectively, to directly measure sympathetic activity. The nerve signal will be
amplified (70,000-fold), bandpass filtered (700-2,000 Hz), rectified, and integrated (time constant 0.1s) using a nerve
traffic analyzer. Nerve signal of MSNA will be confirmed by lack of increase in afferent activity during light stroking of the
skin and a confirmed increase in efferent burst frequency and spontaneous cardiac synchronous efferent bursts during
voluntary end-expiratory apnea.
The cold pressor test is an acute stress-evoking situation that accesses cardiovascular disease risk by measuring the
amplitude of cardiovascular reactivity during the two minutes that the participants hand is submerged in the ice water.
Carroll et al. (2012) showed the greater the amplitude of cardiovascular reactivity to an acute stressor, the lower the
survival rates over the span of 20 years. Beat-to-beat blood pressure derived from finger plethysmography, continuous
ECG derived heart rate, and MSNA activity will be recorded as cardiovascular reactivity for the duration of the cold
pressor test. The greatest change in MSNA burst frequency, heart rate, and blood pressure from baseline measures will
be used for data analysis. Water temperature will be between 0 and 4 degrees Celsius at the time of hand submersion.
Device-guided slow breathing will be completed using the RESPeRATE system set to a respiration rate of 8 breaths/min
for 15 minutes. Participants will be equipped with headphones in which the RESPeRATE system will instruct the
Experimental Visit
participant to inhale and exhale at a time interval congruous with 8 breaths/min. Respiration rate, blood pressure, heart
rate, and MSNA will be recorded for the last 5 minutes of the intervention to ensure compliance and evaluate its
cardiovascular effects. The cold pressor test will be employed during the last 2 minutes of device-guided slow breathing
to assess its effects on cardiovascular reactivity. Data used for analysis will be derived similarly to the “baseline” cold
pressor test. The change score between the “baseline” cold pressor test and the cold pressor test + device-guided slow
breathing will be used for data comparisons.
Mindful meditation will be completed using a pre-recorded 15-minute video. Participants will be equipped with
headphones in which the mindful meditation video will instruct the participant to focus on sensations in the present
moment while accepting their thoughts without judgement. Similar to the device-guided slow breathing intervention,
respiration rate, blood pressure, heart rate, and MSNA will be recorded for the last 5 minutes of the intervention to
ensure compliance and evaluate its cardiovascular effects. The cold pressor test will be employed during the last 2
minutes of mindful meditation to assess its effects on cardiovascular reactivity. Data used for analysis will be derived
similarly to the “baseline” cold pressor test. The change score between the “baseline” cold pressor test and the cold
pressor test + mindful meditation will be used for data comparisons.
Scientific rigor will be upheld by implementing a series of practices in this within-subject design. First, random allocation
of participants into interventions for Experimental Day 1 eliminates an “order effect”. MSNA is the gold-standard for
measuring sympathetic nervous system activity as it is a direct measure of the sympathetic nervous system activity via
the peroneal nerve. Beat-to-beat blood pressure via finger plethysmography is a validated and reliable measure (Schutte
et al., 2004). In addition to its validity and reliability, the Finapres will be re-calibrated to two brachial artery pressures
before each recording period ensuring appropriate calibration before recording data. Data analysts will be blinded as to
which intervention group the data contains. Statistical analyses and sample size has been chosen a priori. Since
participants are not able to be blinded as to which treatment they are receiving, this awareness is an inherit bias that
will be acknowledged within the study findings.
Statistical Analyses
A sample size of 19 will detect a 3 bursts/min difference in MSNA burst frequency with 80% power and a significance
level of 0.05. An additional 6 subjects will be recruited for a total sample size of 25 subjects to account for the 70%
success rate of obtaining an MSNA reading.
The statistical approach for data analysis will use a matched pairs t-test to assess differences in the change score of
MSNA burst frequency from the “baseline” CPT and CPT paired with each breathing intervention. Matched pairs t-tests
will also be used for comparing differences in change scores of respiratory rate, heart rate, blood pressure, and MSNA
burst incidence. Data distributions will be assessed for symmetry; if distributions of variables are not symmetrical, then
data will be compared using non-parametric statistical tests. Data will be reported as means ± standard deviation and
significance will be set at an alpha of 0.05. Lastly, data will be presented as both change scores and absolute values for
transparency.
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