Laboratory
10
Mammalian
Dive Response
Diving bradycardia
in Homo sapiens
Objectives
Your objective today is to run a series of experiments that will uncover the
proximal cues for diving bradycardia.
Introduction
When mammals dive, they must cope with the problem of being denied an external
source of oxygen. Consummate divers such as the remarkable Weddell seal may remain
submerged for up to two hours. To do so, they rely on anatomical features and
physiologicalresponses that increase oxygen storage while reducing the use of
oxygen for nonessential activities during a dive. Blood vessels supplying nonessential
organs are constricted, redirecting blood to the oxygen-requiring brain and
heart. Because it is supplying fewer organs with blood, the heart can beat more
slowly (a condition known as bradycardia) while maintaining adequate blood pressure
to the brain, the most metabolically sensitive organ; a further benefit of bradycardia
is that the heart requires less oxygen as well. Diving bradycardia is an easily
measured component of a group of reflexes that also include holding the breath
(apnea) and peripheral vasoconstriction. Together these reflexes constitute
the "diving response."
In comparison with diving mammals, humans are poorly adaptedto life in the
water. In 2002, free-diving champion Mandy-Rae Cruikshank set a women's world
record for static apnea of 6 minutes 13 seconds (the men's record, set in 2001by
Scott Campbell, is 6 minutes 45 seconds), but most of us are comfortable holding
our breaths for less than a minute.The first part of this laboratory exercise
is designed to demonstrate that, despite our terrestrial nature, humans experience
bradycardia when simulating a dive by holding the breath and immersing their
face in cold water. The second part of this investigatory asks you to consider
the cues that stimulate the bradycardia reflex. This question might be phrased
"how does the body know that it is diving?" As biologists ,we might restate
this question as "what are the proximate signs that trigger diving bradycardia?"
The simulated dive holds several potential proximate cues: 1) apnea and
2) exposure of the face to cold water, which may be further broken down
into several components including coldness, wetness, and pressure. The goal of
the second part of todays exercise is to determine which of these cues triggers
diving bradycardia in humans.
Proceedures
All tests are conducted in the same posture: leaning over the lab bench, headdownwith
elbows resting on the lab bench.
You should work in groups of two or three but we will need the data from the
entire class to tease out small effects with statistical analysis. Each student
takes a turn being the experimental subjece, data collector or data recorder.
Each test lasts 30-60s. Most students will be able to hold your breath this long
without too much trouble but should always stop anytime they experience discomfort.
Test subjects will appreciate being tapped on the back every 10 s by the timer,
particularly when being asked to hold their breath, because it is easy for subjects
to lose track of time during the test.
A few points to remember
- At the beginning of a test that requires holding the breath you should take
a deep but not maximal breath and then hold it
- Do not hyperventilate before holding your breath.
- Before conducting any test using water, you should measure the temperature
of the water and adjust it by adding warm tapwater or ice. Temperature should
be 15°C for the initial simulated dive.
- For immersion tests, subjects should immerse the face up to the temples.
- Allow several minutes for heart rate to return to normal between tests.This
might be the perfect time to switch subjects.
- For temperature tests in air, use a frozen gel pack or plastic freezer bag
filled
with water at 5° C, Below this temperature, cold on the face may cause
an increase in heart rate. The gel packs can be wrapped in a towel, so
they don't feel as cold even if they have been stored in the freezer.
- Between use by different students, basins and snorkels should be disinfected
by immersion in a mild bleach solution (5ml bleach/liter of water) to prevent the spread
of plague and/or cooties.
Equipment Setup
Note: This setup demonstrates basic Lead II ECG connections on a human subject. Student experiments may require variations.\
- Plug the Electrode Lead Set into channel on the MP3 unit.
- Plug the pulse plethysmograph into channel 2
- Turn the acquisition unit on followed by the computer
- Launch the BSL PRO software.
- For channel 1, select the ECG preset from the preset list
- For channel 2, select the plethysmograph preset from the preset list
- The acquisition period will depend on the subjects breath hold ability but 1 to 2 min is usually adiquate to contan the (control preexperiment 30 sec) followed by the experimental data
No Calibration is required but you should collect a bit of data to be certain that the gain for each channel is appropriate. Adjust as required.
Subject — Electrode Connections
- IMPORTANT! Selected subject must not have any known heart conditions.
- Cleane the medial surface of the right and left wrist and the left ankle,with alcohol and abrasive pads.
- Permit the areas to dry then apply gel-filled vinyl electrodes to each area.
- Attach the electrode lead set as follows:
- Right wrist: - White
- Right wrist: - Red
- Left ankle: - Black
Remember that diving bradycardia also includes reduction in blood flow to the extremitise in order to maximize flow to the brain. Total blood flow to the extrematies can be recorded as the area under the curve plotted from the photoelectric plethismograph. Set the plethismograph up on channel 2 and attach the sensor to a finger tip. Do not over tighten the strap.
An additional parameter that may chenge in response to diving is core heat conservation. Changes in the surface temperature of the skin can be monitored with surface thermisters. Thermisters should be mounted with tape on the back of a hand or the top of a foot.
Running the Experiment
Hints for minimizing data error
- The subject should not talk or laugh during any of the recording segments.
- The subject should be relaxed in the position noted for each recording segment.
- The subject should be as still as possible during the recording segment. The electrocardiograph is very sensitive to small changes in voltage caused by contraction of skeletal muscles, and the subject's arms and legs need to be relaxed so that the muscle (EMG) signal does not corrupt the ECG signal.
- Record a 15 to 30 seconds interval to serve as the predive contorl before any experiment.
- Subject should remain immersed for 20-30 seconds — the longer the better!• Screen subjects for smoking and drugs, which alter cardiac activity.
- Prohibit caffeine intake within six hours of the experiment.
- Allow an equilibrium period of no less than 3 minutes between immersions.
Student-designed experiments:
pairs of test conditions for evaluating proximate cues.
After demonstrating that diving bradycardia exists in humans, You should perform
a series of experiments that will uncover the proximate cues that stimulate
the decrease in heart rate. Potential proximate cues can be evaluated with a
series of paired experiments. Note that some variables can be investigated with
more than one set of test conditions; each of these tests yields slightly different
information, and consideration of several experiments togethercan yield further
information not revealed by tests considered separately.
The data
Paired data for each experimental variable and its control allows the use of the very powerful paired t-test. You should record the data (HR) for the 15- to 30-s measurement period priorto each experiment followed immediatly by the experimental data on the same recording . Remember that each paired data set is to be treated as a separate experiment. Making more than
one comparison within an experiment requires more advanced statistical tools—ANOVA
or Bonferroni-corrected t-tests.
In the event that the distribution of the data is significantly nonnormal, the
nonparametric Wilcoxon matched pairs test, which makes no assumptions about
the distribution of the data, should be substituted for the paired t-test.
To make use of paired t-tests, each student must complete both test conditions
in each experiment, and data must be compiled so that pairs of results from
each student are kept together (for instructions on how to format data, see
instructions accompanying your statistical analysis software).
Order effects should be controlled for within each experiment. Use a random number generator to determine the order of the experiments. Flip a coin to determine which of the paired experiments is conducted first.
Data analysis: You need to calculate the frequency of the heart rate in bpm. This can be accomplished automatically by following the instructions in the BioPac manual but the set up is tedious and requires attention to detail. You have only a few data points so manual calculation may be more appropriate.
Set one of the calculations windows to frequency and one to integrate
- Frequency: Enlarge the output so you are looking at the recording for last 15 sec of the control or experimentl data and using the the I beam tool select the interval between each beat for about 10 beats. Record the frequency for each interval and calculate the mean. Remember that BioPac will calculate Htz and you need to convert to BPM. Repeat this for the last 15 sec of each dive oe wexperimen.
- Blood flow: Again enlarge the output so you are looking at about 15 sec of data (control or experimental). Using the I beam tool select the start and end of this 15 second interval. set one of the calculation windows to area and record this number as representitive of flow. This value is in mV/sec and is relative to the set up.Because of this you may need to convert the data to percent change to evaluate the results of the class data.
- The frequency and relative blood flow data can then be analysed statistically. Note: one approach to eliminating the varience in induividuals is to calculate the % change from control to dive. If you do this there is no need to run paired T tests as yo will have only one number for each student for each experiment ( and the entire data set can be analysed woth an ANOVA.
Report
A table presenting the data and its statistical significance and a graphic representation of the results are required along with a paragraph describing the proximate cause of the dive response. If you move through the experiments quickly and everyone enters data the analysis should warent a report. In the event that our n is to low I will add students from previous years.
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Walter I. Hatch
wihatch@smcm.edu
November 18, 2012