The Effects of Nicotine on the The Effects of Nicotine on the Heart Rate of the 5-Day Heart Rate of the 5-Day In In

VitroVitro Chicken Embryo Chicken Embryo

Aaron Kaiser & Daniel Arbeider

Biology 240W

The Pennsylvania State University – Lehigh Valley

PurposePurpose

To evaluate the effects of four different To evaluate the effects of four different dilutions of a 1% nicotine stock solution on dilutions of a 1% nicotine stock solution on the heart rate (bpm) of the 5-day chicken the heart rate (bpm) of the 5-day chicken embryo.embryo.

HypothesisHypothesis

Nicotine will raise the heart rate of Nicotine will raise the heart rate of in in vitrovitro 5-day chicken embryo in 5-day chicken embryo in correlation with dosage.correlation with dosage.

Nicotine exposure will cause cardiac Nicotine exposure will cause cardiac arrest at high doses.arrest at high doses.

Chicken Heart Development (33 Chicken Heart Development (33 hours)hours) The 33 hour chicken embryo The 33 hour chicken embryo

develops four main regions of develops four main regions of the future heart:the future heart:

conotruncus (ct)conotruncus (ct) ventricle (v)ventricle (v) atrium (a)atrium (a) sinus venosus (sv)sinus venosus (sv)

At this time the heart tube At this time the heart tube bends forcing the ventricle bends forcing the ventricle outward.outward.

(McLaughlin and McCain, 1999) (McLaughlin and McCain, 1999)

http://www.lv.psu.edu/jxm57/chicklab/outline.html

Chicken Heart Development (48 Chicken Heart Development (48 hours)hours)

At 48 hours the heart continues At 48 hours the heart continues to bend, forming a single loop.to bend, forming a single loop.

The sinus venosus and atrium The sinus venosus and atrium move behind the ventricle.move behind the ventricle.

(McLaughlin and McCain, 1999) (McLaughlin and McCain, 1999)

http://www.lv.psu.edu/jxm57/chicklab/outline.html

Chicken Heart Development (72 Chicken Heart Development (72 hours)hours)

The atrium expands as it is The atrium expands as it is about to divide into two (left about to divide into two (left and right). and right).

The conotruncus will be the The conotruncus will be the future aorta.future aorta.

(McLaughlin and McCain, 1999) (McLaughlin and McCain, 1999)

http://www.lv.psu.edu/jxm57/chicklab/outline.html

NicotineNicotine

Nicotine is a drug. It Nicotine is a drug. It

acts by mimicking a acts by mimicking a

naturally present naturally present

chemical in the bodies of mammals, chemical in the bodies of mammals, acetylcholine (Vaupel, 2004).acetylcholine (Vaupel, 2004).

Chemical formula- C10H14N2 proper name Chemical formula- C10H14N2 proper name

3-(1methyl-2-pyrrolidinyl)pyridine (Pugh, 3-(1methyl-2-pyrrolidinyl)pyridine (Pugh, 2005). 2005).

Nicotine Structure. New York University

NicotineNicotine

Nicotine mimics the effect of acetylcholine Nicotine mimics the effect of acetylcholine by binding to nicotinic acetylcholine by binding to nicotinic acetylcholine receptors (nAChRs). This triggers the receptors (nAChRs). This triggers the release of adrenaline, causing muscle cells release of adrenaline, causing muscle cells to contract. (Vaupel, 2004). to contract. (Vaupel, 2004).

Nicotine seems to have a localized Nicotine seems to have a localized reaction on the heart, and exposure to reaction on the heart, and exposure to large amounts of nicotine can lead to large amounts of nicotine can lead to cardiac arrest, especially when exercising cardiac arrest, especially when exercising ((Pugh, 2005Pugh, 2005). ).

NicotineNicotine

Because nicotine stimulates the Because nicotine stimulates the release of adrenaline, the heart is release of adrenaline, the heart is constantly being bombarded with constantly being bombarded with signals to speed up. Eventually, signals to speed up. Eventually, the heart may stop acting on the heart may stop acting on these signals, even if more blood these signals, even if more blood needs to be oxygenated during needs to be oxygenated during exercise. Therefore, tissues do not receive enough exercise. Therefore, tissues do not receive enough oxygen and begin to die. If enough heart tissue oxygen and begin to die. If enough heart tissue dies, cardiac arrest can result (Pugh, 2005).dies, cardiac arrest can result (Pugh, 2005).

http://www.biovisuals.com

MethodsMethods

Prepare four serial dilutions, Prepare four serial dilutions, 0.0001%, 0.001%, 0.01%, and 0.1% 0.0001%, 0.001%, 0.01%, and 0.1% nicotine from a 1% nicotine stock nicotine from a 1% nicotine stock solution by diluting it with sterile solution by diluting it with sterile chick saline.chick saline.

““Windowing” an EggWindowing” an Egg

1. “Window” an egg using the methods of Cruz et al., 1993.“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 6. Immediately, obtain in vivoin vivo heart rate (bpm) five times at 15 second heart rate (bpm) five times at 15 second intervals using a stop watch.intervals using a stop watch.

Explanting an EmbryoExplanting an Embryo

1. “Explant” the embryo using the methods of Cruz et al, 1993.1. “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.

6.6. Immediately, obtain Immediately, obtain in vitroin vitro heart rate five times at 15 second heart rate five times at 15 second intervals.intervals.

MethodsMethods

Using a sterile, plastic pipette, remove the saline from the Using a sterile, plastic pipette, remove the saline from the dish and add the smallest concentration of nicotine, dish and add the smallest concentration of nicotine, 0.0001%.0.0001%.

Allow 30 seconds to acclimate to the new solution.Allow 30 seconds to acclimate to the new solution.

Obtain heart rate (bpm) five times at 15 second intervals.Obtain heart rate (bpm) five times at 15 second intervals.

Repeat these steps for the next three solutions of nicotine.Repeat these steps for the next three solutions of nicotine.

ControlControl

The control data was derived The control data was derived from the from the in vivoin vivo and and in vitroin vitro heart rates of the embryo heart rates of the embryo before exposure to the before exposure to the nicotine dilutions.nicotine dilutions.

Chicken Embryo Purdue University

0

50

100

150

200

250

Embryo 1 Embryo 2 Embryo 3 Embryo 4 Embryo 5

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.

0

20

40

60

80

100

120

140

In Vivo In Vitro 0.0001%Solution

0.001%Solution

0.01% Solution 0.1% Solution

Average Heart Rate (bpm)

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.

0

20

40

60

80

100

120

140

160

180

200

In Vivo In Vitro 0.0001% Solution 0.001% Solution 0.01% Solution 0.1% Solution

Embryo 1

Embryo 2

Embryo 3

Embryo 4

Embryo 5Heart

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

  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

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

ResultsResults

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.

ResultsResults

In vivoIn vivo heart rates were generally higher than the heart rates were generally higher than the in vitroin vitro heart rates. Reasons for this may include: heart rates. Reasons for this may include: Shock from the removal of the embryo from its natural Shock from the removal of the embryo from its natural

environment and extraembryonic membranes.environment and extraembryonic membranes. Fluctuations in temperature of the saline solutions Fluctuations in temperature of the saline solutions

throughout experimentation.throughout experimentation. Time constraints for embryo acclimation.Time constraints for embryo acclimation.

When a warmer environment was provided using When a warmer environment was provided using an extra lamp and heating pads, the an extra lamp and heating pads, the in vitro in vitro embryos exhibited heart rates similar to embryos exhibited heart rates similar to in vivo in vivo embryos.embryos.

ResultsResults

In a study conducted by Catherine Sweeney and In a study conducted by Catherine Sweeney and Farouk Markos, and published in Autonomic Farouk Markos, and published in Autonomic Neuroscience, the effects of nicotine on the Neuroscience, the effects of nicotine on the hearts of rats appeared similar to our results. hearts of rats appeared similar to our results. (Sweeney, 2004) (Sweeney, 2004)

ConclusionConclusion

The hypothesis that nicotine raises the The hypothesis that nicotine raises the in vitroin vitro heart rate of heart rate of the 5-day chick embryo in correlation with serial dilutions of the 5-day chick embryo in correlation with serial dilutions of a 1% nictotine stock solution was not supported.a 1% nictotine stock solution was not supported. Though the heart rates did rise initially, they quickly gave way Though the heart rates did rise initially, they quickly gave way

to sporadic arrhythmias.to sporadic arrhythmias.

The hypothesis that high doses of nicotine would lead to The hypothesis that high doses of nicotine would lead to cardiac arrest was supported.cardiac arrest was supported. Shortly after exhibiting specific arrhythmias each embryo Shortly after exhibiting specific arrhythmias each embryo

expired from cardiac arrest.expired from cardiac arrest.

Future ExperimentsFuture Experiments

In future experiments a more stable environment In future experiments a more stable environment should be provided for the explanted embryo.should be provided for the explanted embryo.

Solution increments could be smaller. This would Solution increments could be smaller. This would highlight the threshold at which arrhythmias occur.highlight the threshold at which arrhythmias occur.

The experiment could be conducted directly on the The experiment could be conducted directly on the explanted heart explanted heart in vitroin vitro..

Works CitedWorks Cited

Chadman, K. K. (2004). Cardiovascular effects of nicotine, chlorisondamine, and mecamylamine in the Chadman, K. K. (2004). Cardiovascular effects of nicotine, chlorisondamine, and mecamylamine in the pigeon. pigeon. The Journal of pharmacology and experimental therapeuticsThe Journal of pharmacology and experimental therapeutics, 308(1), 73., 308(1), 73.

Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Press, San Diego, Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Press, San Diego, California, 241 pages. [ISBN 0-12-198390-0] [book]. California, 241 pages. [ISBN 0-12-198390-0] [book].

McLaughlin, D. J. (1999). Developmental and physiological aspects of the chicken embryonic heart. McLaughlin, D. J. (1999). Developmental and physiological aspects of the chicken embryonic heart. Retrieved Mar. 16, 2005, from Chicken Heart Development Lab Web site: Retrieved Mar. 16, 2005, from Chicken Heart Development Lab Web site: http://www.lv.psu.edu/jxm57/chicklab/outline.html. 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: 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. 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: 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.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 Sweeney, C. (2004). The role of neuronal nitric oxide in the vagal control of cardiac interval of the rat heart heart in vitroin vitro. . Autonomic neuroscience, 111Autonomic neuroscience, 111(2), 110.(2), 110.

Vaupel, D. B. (2004). Pharmacological and toxicological evaluation of 2-fluoro-3-(2(s)-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 azetidinylmethoxy)pyridine (2-f-a-85380), a ligand for imaging cerebral nicotinic acetylcholine receptors with positron emission tomography. receptors with positron emission tomography. The Journal of pharmacology and experimental The Journal of pharmacology and experimental therapeuticstherapeutics, 312(1), 355., 312(1), 355.