The Effects of Nicotine on the
Heart Rate of the 5-Day In Vitro
Chicken Embryo
Aaron Kaiser & Daniel Arbeider
Biology 240W
The Pennsylvania State
University – Lehigh Valley
Purpose
To evaluate the effects of four different dilutions
of a 1% nicotine stock solution on the heart rate
(bpm) of the 5-day chicken embryo.
Hypothesis


Nicotine will raise the heart rate of in vitro
5-day chicken embryo in correlation with
dosage.
Nicotine exposure will cause cardiac arrest
at high doses.
Chicken Heart Development (33 hours)

The 33 hour chicken embryo
develops four main regions of the
future heart:





conotruncus (ct)
ventricle (v)
atrium (a)
sinus venosus (sv)
At this time the heart tube bends
forcing the ventricle outward.
(McLaughlin and McCain, 1999)
http://www.lv.psu.edu/jxm57/chicklab/outline.html
Chicken Heart Development (48 hours)

At 48 hours the heart continues to
bend, forming a single loop.

The sinus venosus and atrium
move behind the ventricle.
(McLaughlin and McCain, 1999)
http://www.lv.psu.edu/jxm57/chicklab/outline.html
Chicken Heart Development (72 hours)

The atrium expands as it is about
to divide into two (left and right).

The conotruncus will be the
future aorta.
(McLaughlin and McCain, 1999)
http://www.lv.psu.edu/jxm57/chicklab/outline.html
Nicotine


Nicotine is a drug. It
acts by mimicking a
Nicotine Structure. New York University
naturally present
chemical in the bodies of mammals, acetylcholine
(Vaupel, 2004).
Chemical formula- C10H14N2 proper name 3(1methyl-2-pyrrolidinyl)pyridine (Pugh, 2005).
Nicotine


Nicotine mimics the effect of acetylcholine by
binding to nicotinic acetylcholine receptors
(nAChRs). This triggers the release of
adrenaline, causing muscle cells to contract.
(Vaupel, 2004).
Nicotine seems to have a localized reaction on
the heart, and exposure to large amounts of
nicotine can lead to cardiac arrest, especially
when exercising (Pugh, 2005).
Nicotine

Because nicotine stimulates the
release of adrenaline, the heart is
constantly being bombarded with
signals to speed up. Eventually,
the heart may stop acting on
http://www.biovisuals.com
these signals, even if more blood
needs to be oxygenated during
exercise. Therefore, tissues do not receive enough
oxygen and begin to die. If enough heart tissue dies,
cardiac arrest can result (Pugh, 2005).
Methods

Prepare four serial dilutions, 0.0001%,
0.001%, 0.01%, and 0.1% nicotine from a
1% nicotine stock solution by diluting it
with sterile chick saline.
“Windowing” an Egg
1. “Window” an egg using the methods of Cruz et al., 1993.
2. Place one piece of Scotch tape down the
center of the egg, then one on each
side of that.
3. Using scissors, puncture one end of
the egg and withdraw 1-2 ml of
albumin using the 20G needle.
4. With the scissors, cut an oval shaped opening through the taped section of the
egg.
5. Carefully, remove the shell cap.
6. Immediately, obtain in vivo heart rate (bpm) five times at 15 second intervals
using a stop watch.
Explanting an Embryo
1. “Explant” the embryo using the methods of Cruz et al, 1993.
2. Place a filter paper ring, so-called ‘donut,’ around the
embryo.
3. Using the scissors, cut the extra-embryonic membranes
and blood vessels around the embryo,
detaching them from the egg.
4. Remove the embryo with microsurgical forceps or an
embryo spoon.
5. Place the embryo in a Syracuse dish filled with warm chick saline, then place
dish under a stereomicroscope warmed by a Gooseneck lamp.
6. Immediately, obtain in vitro heart rate five times at 15 second intervals.
Methods

Using a sterile, plastic pipette, remove the saline from the
dish and add the smallest concentration of nicotine,
0.0001%.

Allow 30 seconds to acclimate to the new solution.

Obtain heart rate (bpm) five times at 15 second intervals.

Repeat these steps for the next three solutions of nicotine.
Control

The control data was
derived from the in vivo
and in vitro heart rates of
the embryo before
exposure to the nicotine
dilutions.
Chicken Embryo Purdue University
250
150
100
50
Embryo 2
Embryo 3
Embryo 4
io
n
So
lu
t
0.
1%
ol
ut
S
0.
01
%
0.
00
1%
Embryo 1
io
n
n
S
ol
ut
io
So
lu
tio
0.
00
01
%
Vi
tr
o
In
Vi
vo
n
0
In
Heart Rate (bpm)
200
Embryo 5
Concentration of Nicotine
Figure 1. The change in heart rate (bpm) of five 5-day chicken embryos over
time and exposure to nicotine (concentration used). Control in vivo and invitro
heart rates are also displayed.
140
Heart Rate (bpm)
120
100
80
60
40
20
0
In Vivo
In Vitro
0.0001%
Solution
0.001%
Solution
0.01% Solution
0.1% Solution
Average Heart Rate (bpm)
Concentration of Nicotine
Figure 2. The average change in heart rate (bpm) of five 5-day chicken
embryos over time and exposure to nicotine (concentration used). Control in
vivo and invitro heart rates are also displayed.
200
Heart Rate (bpm)
180
160
140
120
Embryo 1
Embryo 2
100
Embryo 3
Embryo 4
80
Embryo 5
60
40
20
0
In Vivo
In Vitro
0.0001% Solution
0.001% Solution
0.01% Solution
0.1% Solution
Concentration of Nicotine
Figure 3. A bar graph depicting the average change in heart rate (bpm) of five
5-day chicken embryos versus exposure to nicotine (concentration used).
Control in vivo and invitro heart rates are also displayed
Table 1. The Average Heart Rate (bpm) of five 5-day chicken embryos either
exposed, or not-exposed, to nicotine.
Embryo 1
Embryo 2
Embryo 3
Embryo 4
Embryo 5
In Vivo
167.2
135.2
125.6
104
104
In Vitro
133.6
104
124
108
113.6
0.0001% Solution
146.4
92.8
169.6
108.8
109.6
0.001% Solution
159.2
105.6
186.4
30.4
64.8
176
84.8
149.6
0
0
0
142.4
0
0
0
0.01% Solution
0.1% Solution
Results


Nicotine, at the dilutions tested, dramatically raised the in vitro heart rate
of three of the five embryos tested. The two remaining embryos went into
cardiac arrest after the second dilution (0.001%) was applied. A
significant increase in heart rate (bpm) was not observed.
The General Trend:
 In vivo heart rate of the 5-day ‘windowed’ chick embryo appears
stable and high.
 A significant drop in heart rate occurs after explantation of the embryo
to the in vitro situation.
 As nicotine concentrations increase the heart rate increases until a
threshold is reached.
 All of the embryos exposed to nicotine at the dilutions tested suffered
bouts of tachycardia, fibrillations, and eventually cardiac arrest.
Results

In vivo heart rates were generally higher than the in
vitro heart rates. Reasons for this may include:




Shock from the removal of the embryo from its natural
environment and extraembryonic membranes.
Fluctuations in temperature of the saline solutions throughout
experimentation.
Time constraints for embryo acclimation.
When a warmer environment was provided using an
extra lamp and heating pads, the in vitro embryos
exhibited heart rates similar to in vivo embryos.
Results

In a study conducted by Catherine Sweeney and
Farouk Markos, and published in Autonomic
Neuroscience, the effects of nicotine on the
hearts of rats appeared similar to our results.
(Sweeney, 2004)
Conclusion

The hypothesis that nicotine raises the in vitro heart rate of the 5day chick embryo in correlation with serial dilutions of a 1%
nictotine stock solution was not supported.


Though the heart rates did rise initially, they quickly gave way to
sporadic arrhythmias.
The hypothesis that high doses of nicotine would lead to cardiac
arrest was supported.

Shortly after exhibiting specific arrhythmias each embryo expired from
cardiac arrest.
Future Experiments



In future experiments a more stable environment should
be provided for the explanted embryo.
Solution increments could be smaller. This would
highlight the threshold at which arrhythmias occur.
The experiment could be conducted directly on the
explanted heart in vitro.
Works Cited
Chadman, K. K. (2004). Cardiovascular effects of nicotine, chlorisondamine, and mecamylamine in the pigeon. The
Journal of pharmacology and experimental therapeutics, 308(1), 73.
Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Press, San Diego,
[ISBN 0-12-198390-0] [book].
California, 241 pages.
McLaughlin, D. J. (1999). Developmental and physiological aspects of the chicken embryonic heart. Retrieved Mar. 16,
2005, from Chicken Heart Development Lab Web site: http://www.lv.psu.edu/jxm57/chicklab/outline.html.
Pugh, P. (n.d.). What is nicotine?. Retrieved Mar. 15, 2005, from What is Nicotine? Web site:
http://www.galaxygoo.org/nicotine/what_is_nicotine.html.
Pugh, P. (n.d.). How does nicotine act?. Retrieved Mar. 15, 2005, from How Does Nicotine Act? Web site:
http://www.galaxygoo.org/nicotine/what_is_nicotine.html.
Sweeney, C. (2004). The role of neuronal nitric oxide in the vagal control of cardiac interval of the rat heart in vitro.
Autonomic neuroscience, 111(2), 110.
Vaupel, D. B. (2004). Pharmacological and toxicological evaluation of 2-fluoro-3-(2(s)-azetidinylmethoxy)pyridine (2-fa-85380), a ligand for imaging cerebral nicotinic acetylcholine receptors with positron emission tomography. The
Journal of pharmacology and experimental therapeutics, 312(1), 355.