Transcript Investigating the Effects of Varying Concentrations of Epinephrine on 6-Day Chicken Embryos Dan Devine Rachele Cantrel Pandelee Mikroudis ©2007 The Pennsylvania State University.

Investigating the Effects of Varying
Concentrations of Epinephrine on 6-Day
Chicken Embryos
Dan Devine
Rachele Cantrel
Pandelee Mikroudis
©2007 The Pennsylvania State University. All rights reserved.
Purpose


To investigate the effects of
epinephrine on the 6-day chicken
embryonic heart rate (HR; bpm).
Test the effects of different
concentrations of epinephrine to
discover an optimal concentration
that yields safe and dependable
results in increasing the heart rate
(Barry, 1950; Wideman, 1998)
Hypothesis

When subjected to epinephrine, the HR of the 6-day
chicken embryo will increase as per the previous research
of Barry, 1950. Moreover, an optimal concentration will
exist that will induce a reliable and sustainable heart rate;
however, at higher doses arrhythmias will be generated.
www.microscopy-uk.org.uk/.../mlchicken.html
“Fight or Flight”

Set of physiological changes, such as increases in heart
rate, arterial blood pressure, and blood glucose initiated by
the sympathetic nervous system to mobilize body systems
in response to stress.
Epinephrine
http://www.worldofmolecules.com/emotions/epinephrine.htm
Norepinephrine
http://www.worldofmolecules.com/emotions/norepinephrine.htm
Anatomy of the Adrenal Glands
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http://www.allrefer.com/pictures/s1/a0097500-adrenal-gland
Adrenal glands are small,
triangular glands located on
top of both kidneys.
An adrenal gland is made of
two parts: the outer adrenal
cortex and the inner adrenal
medulla.
Adrenal cortex secretes
steroid hormones called
corticosteroids.
Adrenal medulla secretes
catecholamine hormones.
Epinephrine
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Catecholamine hormone
Secreted by the adrenal medulla
Increase the cardiac output and heart rate
Dilate coronary blood vessels
Increase mental alertness
Elevate metabolic rate
http://chem.sis.nlm.nih.gov/chemidplus/ProxyServlet?objectHandle=DBMaint&actionHandle=default&nextPage
=jsp/chemidheavy/ResultScreen.jsp&ROW_NUM=0&TXTSUPERLISTID=000051434
Norepinephrine
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Helps sustain blood pressure
Secreted by the adrenal medulla
Neurotransmitter
Triggers the release of glucose from energy
stores
Increases skeletal muscle readiness
http://chem.sis.nlm.nih.gov/chemidplus/ProxyServlet?objectHandle=DBMaint&actionHandle=default
&nextPage=jsp/chemidheavy/ResultScreen.jsp&ROW_NUM=0&TXTSUPERLISTID=000051412
Methods

Prepare four serial
dilutions, 0.0001g/mL,
0.001g/mL, 0.01g/mL,
and 0.1g/mL
epinephrine from a
1g/mL stock solution by
diluting it with CMRL
stock media.
Methods
“Window” a 6 day chicken
embryo using the methods
of Cruz, 1993:

Place scotch magic tape on
horizontal end of the egg in
dish lined with cotton.

Carefully cut a “window” in
the eggshell.
Methods

Insert a needle and withdraw
1-2 mL of albumen.

Take the in vivo heart rate of
embryo by counting the
number of heart beats in a
15 second interval. Repeated
this four times, then average.
Methods
“Explant” the six-day old chicken embryo
using the methods of Cruz, 1993.
 Place paper ‘doughnut’ on blastoderm
so it frames embryo, then wait for
vitelline envelope to adhere.
 Cut vitelline envelope along edge of
doughnut with fine surgical scissors.
 Use forceps to quickly transfer
explanted embryo to Syracuse dish
containing CMRL under the
stereoscope with heat lamps.
 Determine in vitro heart rate of chicken
embryo by counting the number of
heart beats in a 15 second interval.
Repeat this four times, then average.
Methods

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Explant the heart using the
microdissecting scissors and a pair
of fine forceps.
Position the embryo so that the
left side is facing upward.
Use the microdissecting scissors
and a pair of fine forceps, remove
the left thorax from the embryo.
Using both pairs of forceps,
carefully remove the heart from
the surrounding tissues. Discard
all tissue except for heart itself.
Determine the in vitro heart rate
of the isolated heart by counting
the number of heart beats in a 15
second interval. Repeated this
four times, then average.
Methods


Use the “building-up,”
method, defined herein, to
gradually increase the
concentration of epinephrine
that the embryo is exposed
to.
Record the heart rate after
each increase in drug
concentration.
Results
140
Beats Per Minute
120
100
80
60
40
20
0
In vivo
In vitro
Explanted Heart
Condition
Figure1: Histogram showing the average heart rates (bpm) of embryos
prior to exposure to epinephrine (Control data).
Results
90
80
Beats Per Minute
70
60
50
40
30
20
10
0
0.0001
0.001
0.01
0.1
Epinephrine Concentration
Figure2: Histogram showing the average heart rates (bpm) for in vitro embryos
exposed to various concentrations of epinephrine.
Results
120
Beats Per Minute
100
80
60
40
20
0
0.0001
0.001
0.01
0.1
Epinephrine Concentration
Figure3: Histogram showing the average heart rates (bpm) for explanted hearts
exposed to various concentrations of epinephrine.
Results
160
140
Beats Per Minute
120
Embryo 1
Embryo 2
100
Embryo 3
Embryo 4
80
Embryo 5
Embryo 6
60
Embryo 7
Embryo 8
40
Embryo 9
20
0
In vivo
In vitro
Exp.
Heart
0.0001
g/mL
0.001
g/mL
0.01
g/mL
0.1 g/mL
Firgure4: Histogram comparing the heart rates (bpm) of the nine
experimental embryos.
Conclusion
•The hypothesis that an optimal concentration will exist and will induce a reliable and
sustainable heart rate was supported.
•Data gathered supports an optimal epinephrine concentration of 0.01 g/mL solution
•The hypothesis that at higher doses of epinephrine, arrhythmias will be generated was
supported.
•Data gathered supports the generation of atrial fibrillation, atrial flutter, and ventricular
flutter at 0.1 g/mL epinephrine.
90
80
120
100
60
Beats Per Minute
Beats Per Minute
70
50
40
30
80
60
40
20
20
10
0
0
0.0001
0.001
0.01
Epinephrine Concentration
0.1
0.0001
0.001
0.01
0.1
Epinephrine Concentration
Figure5: Comparison of histograms from Figure2 (left) and Figure3 (right) showing
similar trends in heart rates (bpm)
Further Experiments
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Collect Data from a larger number of embryos.
Use more controlled environments for the explanted
embryos to stabilize temperature.
Pinpoint, more precisely, the exact optimal
concentration by testing serial dilutions surrounding
0.01 g/mL epinephrine in more defined increments.
Conduct in vivo experiments on older, more developed,
embryos by using a time-lapse video to record heart
rates of embryos following injection of drug through
the shell and extra-embryonic membranes.
References
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Barry, A. (1950).The effect of epinephrine on the myocardium of the embryonic chick.
Journal of the American Heart Association. 1, 1362-1368.
Campbell, Neil A., & Reece, Jane B. (2005). Biology (7th ed.).
San Francisco: Benjamin Cummings.
Cooke, K. V. (2005). Epinephrine. Retrieved March 20, 2007, from WebMD Web site:
http://www.webmd.com/hw-popup/Epinephrine
Cruz, Y. P. 1993. Laboratory exercises in developmental biology. Academic Press, San Diego,
California, 241 pages. [ISBN 0-12-198390-0] [book]
Iverson, Carlyn (2007, April 9). Adrenal Gland. Retrieved April
9, 2007 from , Web site: http://www.allrefer.com/pictures/s1/a0097500-adrenalgland
McLaughlin, J.S. and McCain, E.R. (1997) "In Vivo and In Vitro Development of
the Chicken Heart."Tested Studies for Laboratory Teaching, Volume 19 (C.A. Goldman,
Editor). Proceedings of the 19th Workshop/Conference of the Association for
Biology Laboratory Education (ABLE) 19: 331-332.
Wideman, R. F. (1998).Cardiac output in four-, five-, and six-week-old broilers, and
hemodynamic responces to intravenous injections of epinephrine. Poultry Science. 78,
392–403.
Special Thanks To:
Dr. Jacqueline McLaughlin
Joey Mamari (Moral Support)
Lynn Abeln
Michele Hoback
Samer Moussa