The%20Effects%20of%20Nicotine%20on%20the%20Heart%20Rate%20of%20the%205-Day%20In%20Vitro%20Chicken%20Embryo

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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
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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.

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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.

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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
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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
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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
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Nicotine

• Nicotine is a drug. It
• acts by mimicking a
• naturally present
• chemical in the bodies of mammals, acetylcholine
  (Vaupel, 2004).
• Chemical formula- C10H14N2 proper name
  3-(1methyl-2-pyrrolidinyl)pyridine (Pugh, 2005).

Nicotine Structure. New York University
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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).

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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
• 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).

http//www.biovisuals.com
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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.

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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.

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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.
• Place the embryo in a Syracuse dish filled with
  warm chick saline, then place dish under a
  stereomicroscope warmed by a Gooseneck lamp.
• Immediately, obtain in vitro heart rate five
  times at 15 second intervals.

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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.

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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
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Heart Rate (bpm)
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.
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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.
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Heart Rate (bpm)
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
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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
0.01 Solution 176 84.8 149.6 0 0
0.1 Solution 0 142.4 0 0 0
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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.

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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.

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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)

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Conclusion

• The hypothesis that nicotine raises the in vitro
  heart rate of the 5-day 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.

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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.

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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,
  California, 241 pages. ISBN 0-12-198390-0
  book.
• 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/w
  hat_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)-azeti
  dinylmethoxy)pyridine (2-f-a-85380), a ligand for
  imaging cerebral nicotinic acetylcholine
  receptors with positron emission tomography. The
  Journal of pharmacology and experimental
  therapeutics, 312(1), 355.