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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. Purpose.

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the effects of nicotine on the heart rate of the 5 day in vitro chicken embryo

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
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
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
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
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
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
  • 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

nicotine8
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).
nicotine9
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

methods
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
“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.

slide12

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.
methods13
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
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

slide15

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.

slide16

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.

slide17

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

slide18

Table 1. The Average Heart Rate (bpm) of five 5-day chicken embryos either exposed, or not-exposed, to nicotine.

results
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.
results20
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.
results21
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
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.
future experiments
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
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/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-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.