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THE EFFECTS OF PHENYLEPHRINE, NITROPRUSSIDE, AND

HYPOXIA ON THE AND VESSELS IN DANIO RERIO

A Thesis

Presented to

The Graduate Faculty at the University of Akron

In Partial Fulfillment

of the Requirements of the Degree

Master of Science

Dakota Turner

December, 2016

THE EFFECTS OF PHENYLEPHRINE, SODIUM NITROPRUSSIDE, AND

HYPOXIA ON THE HEART AND BLOOD VESSELS IN DANIO RERIO

Dakota Turner

Thesis

Approved: Accepted:

______Advisor Dean of the College Dr. Brian Bagatto Dr. John Green

______Committee Member Dean of the Graduate School Dr. Richard Londraville Dr. Chand Midha

______Committee Member Date Dr. Rolando JJ Ramirez

______Department Chair Dr. Steve Weeks

ii ABSTRACT

Pharmaceutical medications in waterways are a growing problem worldwide.

These include antibiotics, heart medications, and artificial steroids. We examined how a pair of commonly prescribed pharmaceuticals, phenylephrine and sodium nitroprusside, affected the heart and blood vessels in developing zebrafish, Danio rerio. The were exposed from 1 day post fertilization (dpf) to 6 dpf. Half of each treatment group was exposed to acute hypoxia on 8 dpf and 15 dpf. On 7 dpf, 8 dpf, 14 dpf, and 15 dpf, , end diastolic volume, end systolic volume, volume, , arterial diameter, and venous diameter were examined via inverted microscope. My results support the concern that pharmaceutical agents can cause physiological changes in locally exposed flora and fauna. Phenylephrine decreased heart rate and in the and hypoxia caused a decrease in heart rate and in the .

iii TABLE OF CONTENTS

Page

LIST OF FIGURES ...... vi

CHAPTER

I. INTRODUCTION ...... 1

Pharmaceuticals in the Waterways ...... 1

Developmental Windows and Deviations ...... 3

Effects on Vasculature by Phenylephrine and Sodium Nitroprusside ...... 3

Environmental Stresses ...... 6

Zebrafish as a Model...... 7

Aims ...... 8

Hypotheses ...... 8

II. MATERIALS AND METHODS ...... 10

Animals ...... 10

Chemical Treatment ...... 11

Acute Hypoxia Exposure ...... 11

Video Data Collection ...... 12

ANOVA ...... 13

III. RESULTS ...... 15

Heart Rate ...... 15

End Systolic Volume and End Diastolic Volume ...... 18

iv ...... 21

Cardiac Output ...... 23

Artery Diameter ...... 25

Vein Diameter ...... 27

IV. DISCUSSION ...... 30

Chronic Phenylephrine Exposure Effects ...... 30

Chronic Sodium Nitroprusside Exposure ...... 32

Acute Hypoxia Exposure ...... 34

Conclusions ...... 36

Final and Future Directions ...... 36

REFERENCES ...... 38

v LIST OF FIGURES

FIGURE Page

1 Timeline of experiment ...... 14

2 Mean  standard error of heart rates in Danio rerio ...... 16

3 Mean  standard error of heart rates in Danio rerio ...... 17

4 Mean  standard error of end systolic volume in Danio rerio...... 18

5 Mean  standard error of end systolic volume in Danio rerio...... 19

6 Mean  standard error of end diastolic volume in Danio rerio ...... 20

7 Mean  standard error of end diastolic volume in Danio rerio...... 21

8 Mean  standard error of stroke volume in Danio rerio ...... 22

9 Mean  standard error of stroke volume in Danio rerio ...... 23

10 Mean cardiac output of untreated, Phe treated, and SNP treated Danio rerio...... 24

11 Mean  standard error of cardiac output in Danio rerio ...... 25

12 Mean  standard error of tail diameters in Danio rerio...... 26

13 Mean  standard error of tail artery diameters in Danio rerio...... 27

14 Mean  standard error of tail diameters in Danio rerio ...... 28

15 Mean  standard error of tail vein diameters in Danio rerio ...... 29

vi CHAPTER I

INTRODUCTION

In the late 1990’s and early 2000’s, wastewater from cities and towns started gaining attention because it contained measurable amounts of pharmaceutical agents

(Grossberger et al., 2014, Bunch et al., 2011, Rocco et al., 2010). While most pharmaceuticals can be removed by water treatment technologies prior to consumption, the wastewater treatment process does not completely prevent the continuing introduction of pharmaceutical agents into waterways (Rocco et al., 2010).

These pharmaceuticals include but are not limited to , antibiotics, anti- inflammatories, , and vasodilators (Grossberger et al., 2014). are one example of many species that live in waters contaminated with various pharmaceuticals.

Therefore, there are concerns about the physiological impact(s) on fish development and the ability to respond to stress.

Pharmaceuticals in the Waterways

Humans use a variety of pharmaceuticals every day, including antibiotics, painkillers, high medication, opioids, anti-, and artificial (Grossberger et al., 2014; Bunch et al., 2011; Rocco et al., 2010).

Pharmaceuticals are expelled from the intact or as derivatives and eventually make their way into the environment (Grossberger et al., 2014; Bunch et al., 2011; Rocco et al., 2010). The most commonly discussed pharmaceuticals in the environment are 1 antibiotics, but other pharmaceuticals, such as artificial hormones, are garnering more attention because of bioaccumulation concerns (Grossberger et al., 2014).

Bioaccumulation of pharmaceuticals occurs in the local fauna and can cause permanent physiological changes in animals (Grossberger et al., 2014; Bunch et al., 2011;

Rocco et al., 2010). The wide use and variety of pharmaceuticals in human populations has had lasting impacts on the local plant and life already. Artificial estrogens are chemical compounds that imitate the female , commonly used in artificial birth control (Johnson et al., 2008). The presence of endocrine-disrupting artificial estrogens in the environment has caused developmental feminization of male fish of many species, including those in the Puget Sound. Bioaccumulation of artificial estrogens in the male fish will cause their to produce vitellogenin, a protein typically produced by female fish and found in egg yolks, leading to a host of reproductive issues including production of reproductive structures with male and female germ cells and decreased sperm count (Johnson et al., 2008). Another pharmaceutical commonly found in the environment, sildenafil citrate, is a vasodilator commonly sold and used under the brand name Viagra (Rocco et al., 2010). Sildenafil citrate induces

DNA degradation and genetic defects that can cause cancer, genetic aberrations, and other genetic issues in zebrafish (Rocco et al., 2010). These few examples illustrate that the presence of human released pharmaceuticals like estrogens and sildenafil citrate have on local fauna is a growing concern because we do not yet know the extent of their impact (Nieto et al., 2010).

2 Developmental Windows and Deviations

Development is the process of growth from egg to juvenile to adult. Every species has a specific pattern of development created by a timed cascade of gene expression, but there is variation. Individuals can alter their developmental trajectory in response to environmental factors such as increased predation, altered temperature, or lack of food.

For example, several species of Daphnia, a small water plankton, will form spiked helmets in response to predator cues in the environment (Enteman, 1900). These helmets will help protect the Daphnia from predation but become a permanent phenotypic alteration changing body size and shape (Enteman, 1900). Conversely, there are examples where environmental factors (natural or artificial) do not affect developmental trajectory.

This may be because the cue is not strong enough or it did not occur during a sensitive developmental stage. Work by Pelster et al. (2005) studying chronic phenylephrine exposure in zebrafish showed a significant increase in heart rate 4 dpf, but no significant difference in heart rate on any other day of analysis despite chronic treatment. This means that some cues must occur at very specific times in sufficient quantities to have a chronic effect.

Effects on Vasculature by Phenylephrine and Sodium Nitroprusside

Sildenafil citrate is a vasodilator commonly found in the aquatic environment

(Rocco et al., 2010; Grossberger et al., 2014). It is a pharmaceutical used to treat erectile dysfunction by vasodilating blood vessels. Sildenafil citrate functions by preventing the breakdown of cyclic GMP groups, creating an excess of cyclic GMP and creating vasodilation (Maharaj et al., 2009). Sildenafil-caused vasodilation is an acute response.

3 Chronic exposure responses to sildenafil citrate are not well documented. To measure chronic effects of vasodilation, we will use an available vasodilator, sodium nitroprusside, because it utilizes the same mechanism of vasodilation as sildenafil citrate.

Because vasoconstrictors are also a commonly prescribed pharmaceutical and have the exact opposite effect of vasodilators, we will also use phenylephrine to determine the effect of chronic vasoconstrictor exposure. Previous experiments have analyzed the effects of phenylephrine or sodium nitroprusside (Naida et al., 2009; Moore et al., 2006;

Lim et al., 2011; Pelster et al., 2005; Fritsche et al., 2000). Most previous studies are focused on specific variables, including heart rate (Naida et al., 2009; Pelster et al.,

2005), -adrenergic receptors (Moore et al., 2006) lacunar (Lim et al., 2011), cardiac output (Pelster et al., 2005), and arteries (Fritsche et al., 2000). While each of these structures have been analyzed separately, one experiment analyzing multiple variables concurrently could analyze how phenylephrine or sodium nitroprusside effects the cardiovascular system under chronic exposure.

Vasoconstrictors, like vasodilators, are commonly prescribed pharmaceuticals.

While a vasodilator, sildenafil citrate, has been commonly found in environmental water samples, vasoconstrictors have not been (Grossberger et al., 2014; Rocco et al., 2010).

Because sildenafil citrate is one of many vasodilator pharmaceuticals and there are many vasoconstrictor pharmaceuticals, we decided to analyze chronic exposure of a vasoconstrictor as well. Phenylephrine is a vasoconstrictor that is used to treat ailments such as and the common cold. Phenylephrine mimics the shape and function of noradrenaline by binding to alpha-adrenergic receptors, causing (Broadley, 2010; Naida et al., 2009; Xu et al., 2011). In Synbrachus

4 marmoratus, the swamp eel, injected phenylephrine after 20 seconds caused a significant decrease in stroke volume and cardiac output, but a significant increase in , dorsal , and mean circulatory filling pressure (Skals et al., 2006).

It had no significant effect on the heart rate (Skals et al., 2006). However, in zebrafish, concentrations of phenylephrine as low as 10 M in the water can cause increased heart rate in zebrafish of about 20 bpm (Naida et al., 2009). Most studies involving phenylephrine in zebrafish are acute exposures followed by study of melanin accumulation or protein production (Xu et al., 2011; Steele et al., 2011; Naida et al.,

2009). In this study, we use phenylephrine as a chronic vasoconstrictor to analyze the effects on the heart and blood vessels.

First used to treat via vasodilation in 1955, sodium nitroprusside has been used in medical treatments for many years (Verner, 1974). Sodium nitroprusside works by producing excess nitric oxide (Pelster et al., 2005). The nitric oxide then enters the guanylate cyclase pathway and produces vasodilation. Vasodilation was first observed in the kidneys in both arteries and veins, but has been observed in the duodenum, uterus, and blood vessels (Verner, 1974; Pelster et al., 2005; Fritsche et al.,

2000). Zebrafish chronically exposed to sodium nitroprusside had a significant decrease in stroke volume after 8 dpf and was severely reduced at 12 dpf (Pelster et al., 2005).

Zebrafish under chronic sodium nitroprusside exposure also develop a reduced rate of consumption (Pelster et al., 2005) There is evidence that sodium nitroprusside treatment around 5 dpf may permanently decrease heart rate in developing zebrafish

(Bagatto, 2005; Pelster et al., 2005). Because sodium nitroprusside produces a

5 vasodilating effect like sildenafil citrate, it is a good pharmaceutical to test for chronic effects on the heart and vasculature in this study.

The concern of chronic effects of environmental pharmaceuticals, such as sildenafil citrate, is how they can change the phenotype of local flora and fauna. These changes may seem minute, but because of the large number of natural environmental stresses that exist, anything that permanently alters the phenotype could have an impact on stress response.

Environmental Stresses

Aquatic systems have a variety of natural stresses (Riou et al., 2012; Allinson et al., 2011; Lares et al., 2012). These include but are not limited to extremes in temperature

(Thorpe et al., 1994; Barrionuevo et al., 1999), salinity (Thorpe et al., 1994), hypoxia

(Burt et al., 2012; Marks et al., 2005), and water flow rate (Bagatto et al., 2001).

Hypoxia is a particularly concerning environmental stress because oxygen is a necessity for most multicellular and localized hypoxic conditions in aquatic environments can be caused by a variety of factors. Hypoxia is the reduction of available

oxygen in the water and is defined as O2<5 mg/l for mild hypoxia to O2>1 mg/l for severe hypoxia (Perry et al., 1992; Pasha et al, 2010). Hypoxia affects the heart by decreasing the available oxygen for oxidative phosphorylation in mitochondria, reducing ATP production and a decrease in heart rate (Kang et al., 2000). Many fish species have developed genetic responses, such as Hif-1 alpha, to hypoxia that cause temporary changes in heart rate, increase or decrease of blood vessel dilation, and reduced growth rate (Ton et al., 2003; Pasha et al., 2010; Zhu et al., 2011; Bagatto, 2005; Moore et al.,

6 2006). Hif-1 alpha induces production of NO synthase, which produces nitric oxide that enters the guanylyl cyclase pathway, leading to blood vessel dilation (Jung et al., 2000).

Chronic hypoxia exposure during development in zebrafish causes a decrease in development rate, a decrease in heart rate, and vasodilation in the dorsal artery (Bagatto,

2005; Marks et al., 2005). Because hypoxia creates changes in blood vessel diameter and has several secondary effects on the heart, it is a good stress to see how the chronic pharmaceutical treatments could be affected by acute hypoxia exposure.

Zebrafish as a Model

Danio rerio, also known as zebrafish, are a common species of fish used in laboratories and sold in pet stores. They produce large clutches allowing for population studies and the individual stages of development have been recorded in great detail

(Bradford et al., 2011; Kimmel et al., 1995). At early stages, the fish is nearly transparent, allowing visualization of the internal organs and blood cells (Kimmel et al.,

1995). Certain morphs, including blonde, Casper, and albino, have delayed or reduced aggregation of pigments so their internal organs can be visualized relatively later in development (Fritsche, 2000).

7 Aims

1. Determine how chronic exposure to phenylephrine and sodium nitroprusside

during early development of zebrafish affects the function of the heart and

vasculature in later development

2. Determine how chronic exposure to phenylephrine and sodium nitroprusside

affects response to acute hypoxia exposure

Hypotheses

1. Chronic phenylephrine treatment will cause vasoconstriction in the arteries

and veins. The heart will respond by increasing heart rate, stroke volume, and

cardiac output. These effects will happen during early development. After

treatment removal the fish will maintain the phenylephrine phenotype on the

heart and vasculature. Sodium nitroprusside will affect the same systems but

will cause vasodilation, slowed heart rate, lower stroke volume, and lower

cardiac output. The treatments will occur over specific developmental

windows, and we predict they will lead to permanent physiological changes

2. Acute (short-term) hypoxia exposure will cause vasodilation, decreased heart

rate by O2 deprivation, and decreased stroke volume via. The effects of acute

hypoxia exposure will be modified by the earlier pharmaceutical treatments.

Phenylephrine treatment will antagonize the effects of acute hypoxia

exposure, but sodium nitroprusside treatment with have either no effect on

acute hypoxia exposure or they will combine to produce a greater amount of

vasodilation, decreased heart rate, and decreased stroke volume. We predict

phenylephrine and acute hypoxia will react as stated because they have

8 competing effects on the heart and veins while sodium nitroprusside and acute hypoxia have complimentary effects.

9 CHAPTER II

MATERIALS AND METHODS

Animals Blonde adult zebrafish were purchased from Two Pet Shop (Akron, Ohio) and Pet Supplies Plus (Stow, Ohio) and used as breeding stock for experiments. Fish were housed in a 95-liter community tank with return water treated mechanically, biologically, and ultraviolet light sterilized with a 14L:10D light cycle. Breeding procedures were followed according to Bagatto et al. (2001). Briefly, a breeding box with artificial grass was placed in the tank at night. The breeding box was removed one hour after the lights turned on in the morning.

Eggs were collected at one hour post-fertilization (hpf) and placed in a beaker in an incubator at 27.0C  0.5. At 24 hpf, viable eggs were randomly sorted into 500-ml treatment beakers of 50 eggs per beaker for each treatment. A total of 506 fish were in this study.

Larvae were fed twice daily starting at 4-5 days post fertilization (dpf) and continued until 15 dpf. Water changes (see below in treatments under subsection phenylephrine) were performed daily along with waste and excess food removed. All experiments were approved by the University of Akron IACUC committee (protocol 11-

9D).

10 Chemical Treatment

Eggs were collected at 24 hpf from the incubator and placed into 500-ml treatment beakers. Each beaker held 300 ml of water and 50 eggs from 24 hpf to 6 dpf.

Three milliliters of water was removed and replaced with 3.0 ml of 1 x 10-4 M phenylephrine solution for a final concentration of 1 x 10-6 M phenylephrine (Naida et al.,

2009; Fritsche et al., 2000). At 24 hpf and continuing daily to 6 dpf, water was drained to

50 ml in the beaker and refilled with 247 ml of water and 3 ml of phenylephrine solution in order to maintain a consistent exposure concentration. At 7 dpf, phenylephrine exposure ceased.

The sodium nitroprusside treatment, exposure concentration = 1 x 10-6 M, followed the same protocol as the phenylephrine treatment.

Acute Hypoxia Exposure

At 8 dpf and 15 dpf, fish from untreated, phenylephrine, and sodium nitroprusside treatments were each split further into two groups. One group was placed in hypoxic tanks with the other group remained in normoxic tanks. Hypoxic water was maintained by bubbling nitrogen (N2) to lower the dissolved oxygen to 0.8  0.5 mg O2/L (Marks et al., 2005). After the oxygen concentration reached 0.8 mg O2/L, the N2 bubbler was removed, the beaker was covered, and the fish were exposed to the hypoxic water for one hour. After one hour, fish were randomly selected and placed in agarose gel for microscope viewing (see video data collection). After microscope examination, fish were returned to normoxic tanks for the remainder of day 8 until day 15. Any fish that were exposed to hypoxia but not examined were also returned to normoxic conditions. A

11 maximum of three hours of acute hypoxia exposure was allowed before the water was restored to normoxic conditions. To ensure there were no side effects from longer hypoxia exposure, collected data was analyzed for a trend difference and compared to time stamps from video collection. Normoxic water was maintained using air bubblers to maintain O2 levels.

Video Data Collection

At 7 dpf, 8 dpf, 14 dpf, and 15 dpf, up to 25 fish were selected from each treatment and viewed under an inverted microscope. For immobilization, a solution of

1% SeaPlaque low melt-agarose gel was prepared and maintained at 40C (Bagatto et al.,

2006; Schwerte et al., 2000). The larva were removed from the beaker with a 5-ml pipette and placed on an agarose bed and covered with agarose gel. After one minute, the larva were placed in a viewing mount and placed on a stage heated to 27C for heart and tail visualization. To collect measurements of the heart, an outline of the and length across widest part of the heart were measured during the and of a heart cycle. This was repeated five times per larva and the data was input into the equation

((8/(3))*A2)/L to determine volume. These morphological means were then used to calculate the stroke volume. The video was analyzed frame by frame from diastole to systole to determine heart rate. This was repeated 5 times and averaged to determine heart rate.

The imaging technique used in this study is based on previous work by Bagatto et al. (2001; Schwerte et al., 2000). An inverted microscope (Leica DMRB) equipped with a

Motionscope PCI 2000 S camera was used to visualize the heart and vessels. Image

12 capture was performed using ImagePro 4.5 software and captured at 120 frames per second for a period of 10 seconds.

The method to record arterial and venous diameter was originally from Fritsche et al. (2000) and applied to examine the main axial artery and axial vein instead of the caudal tail loop. The axial artery and axial vein were located in the recorded frames and a line was drawn across to collect the diameter. This was repeated 5 times at each vessel and averaged.

ANOVA

A three-way ANOVA was performed to analyze the effects of pharmaceutical treatments, acute hypoxia exposure, and development time on the variables measured.

The drug exposure (no drug, phenylephrine, sodium nitroprusside) was the first variable, the oxygen condition (hypoxia, normoxia) was the second variable, and the day of observation was the third variable. The 7 structures analyzed included: heart rate, cardiac output, end diastolic volume, end systolic volume, stroke volume, arterial diameter, and venous diameter. If ANOVA analysis determined a significant difference, post hoc

Tukey’s tests analyzed the data points to determine significant differences between treatments by taking the means of the first variable minus the means of the second variable and dividing the answer by the standard error. The produced q-value was compared to a studentized range distribution and any q-value higher than the table value was significant.

13

Group 1

Group 2

Group 3

Day

Figure 1. Timeline of events. Starting at 1 day post fertilization (dpf), the fish were randomly sorted into one of three groups: untreated, chronic phenylephrine, or chronic sodium nitroprusside. At 6 dpf, all chronic treatments stopped. On 7, 8, 14, and 15 dpf fish from each treatment group were examined via microscope. On 8 and 15 dpf fish from each treatment were put under acute hypoxia exposure for 1 hour before microscope examination.

14 CHAPTER III

RESULTS

Heart Rate

Heart rate was examined for changes caused by pharmaceutical treatment and acute hypoxia exposure. Figure 2 shows the effect of the pharmaceutical treatment on heart rate. Sodium nitroprusside exposure did not have a significant effect on heart rate, but chronic exposure to phenyleprhine had an interaction effect during certain days that caused a significant decrease in heart rate (P=0.0191). Post hoc Tukey analysis of phenylephrine-treated fish revealed a significant decrease in heart rate on days 8 and 14 compared to day 7, but on day 15 heart rate returned to day 7 values.

Under acute hypoxia exposure, phenylephrine exposed fish had a significantly decreased heart rate compared to day 15 but not to day 8 (P=0.0371; Fig. 3). There was also a significant decrease in heart rate between untreated acute hypoxia fish and sodium nitroprusside acute hypoxia fish on day 15.

15

200 29 14 5 31 24 7 19 20 16 11 8 3 180 * * 160 140 120 Untreated 100 Phe 80 SNP 60 40 20 Heart rate (beats perminute)Heart rate 0 7 8 14 15 Days Post Fertilization

Figure 2. Mean  standard error of heart rates in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All treatments stopped at 7 dpf and the were examined via microscope at 7, 8,

14, and 15 dpf. Asterisks mark values significantly different (P<0.05) from day 7 values within each drug treatment group as determined by Tukey’s test. The number of individual measurements in each group are marked above each bar.

16 200 31 25 24 7 24 1 11 7 9 3 8 11 180 160 * Untreated Normoxia 140  Untreated Hypoxia 120 Phe Normoxia 100 Phe Hypoxia 80 60 SNP Normoxia 40 SNP Hypoxia 20 Heart Heart Rate (beatsper minute) 0 N H N H N H N H N H N H 8 8 15 15 Days Post Fertilization

Figure 3. Mean  standard error of heart rates in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposures stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to acute hypoxia (H) or left in normoxia (N) for one hour before examination via microscope. The number of individual measurements in each group are marked above each representative bar. An asterisk denotes a significant difference caused by acute hypoxia exposure compared to normoxia within the respective drug treatment. The  denotes a significant difference between drug treated and untreated fish during acute hypoxia exposure.

17 End Systolic Volume and End Diastolic Volume

Measurements of end systolic volume (esv) and end diastolic volume (edv) were collected to examine pharmaceutical treatment effects on heart function. Treatments of phenylephrine and sodium nitroprusside had no significant effect on esv or edv

(P=0.8343; figure 4 for esv, P=0.9156; figure 6 for edv). Acute hypoxia exposure had no effect on esv or edv (P=0.8627; figure 5 for esv; figure 7 for edv).

0.014 12 7 4 17 16 4 11 15 9 9 2 5 0.012

0.01

0.008 Untreated Phe 0.006 SNP 0.004

0.002 End Systolic Volume (nl)SystolicEnd Volume 0 7 8 14 15 Days Post Fertilization

Figure 4. Mean  standard error of end systolic volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposures stopped at 7 dpf and the hearts were examined via microscope at 7, 8, 14, and 15 dpf.

18

0.014 17 14 13 4 16 1 9 4 2 2 5 5 0.012 Untreated Normoxia 0.01 Untreated Hypoxia 0.008 Phe Normoxia 0.006 Phe Hypoxia SNP Normoxia 0.004 SNP Hypoxia

0.002 End Systolic Volume (nL)SystolicEnd Volume 0 N H N H N H N H N H N H 8 15 Days Post Fertilization

Figure 5. Mean  standard error of end systolic volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to hypoxia (H) or left in normoxia (N) for one hour before examination via microscope.

19

0.025 12 7 4 17 16 4 11 15 9 9 2 5

0.02

0.015 Untreated Phe 0.01 SNP

0.005 End DaistolicEndVolume (nl) 0 7 8 14 15 Days post Fertilization

Figure 6. Mean  standard error of end diastolic volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposures stopped at 7 dpf and the hearts were examined via microscope at 7, 8, 14, and 15 dpf.

20 0.025

12 17 7 16 4 4 11 9 15 5 9 2 0.02 Untreated Normoxia Untreated Hypoxia 0.015 Phe Normoxia Phe Hypoxia 0.01 SNP Normoxia

0.005 SNP Hypoxia End DiastolicEndVolume (nL) 0 N H N H N H N H N H N H 8 15 8 15 Days Post Fertilization

Figure 7. Mean  standard error of end diastolic volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug treatments stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to acute hypoxia (H) or left in normoxia

(N) for one hour before examination via microscope.

Stroke Volume

Stroke volume was calculated from difference between end diastolic volume and end systolic volume. Figure 8 shows how pharmaceutical treatment affected stroke volume over the course of the experiment. There were no significant differences caused by any pharmaceutical treatments (P=0.8286). Figure 9 shows acute hypoxia exposure did not have a significant effect on stroke volume in any treatment group (p=0.7797).

21 0.009 0.008 12 7 4 16 16 4 11 15 9 10 5 2 0.007 0.006 Untreated 0.005 Phe 0.004 SNP 0.003

Stroke VolumeStroke (nl) 0.002 0.001 0 7 8 14 15 Days post Fertilization

Figure 8. Mean  standard error of stroke volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf and the hearts were examined via microscope at 7, 8, 14, and 15 dpf.

22 0.01 0.009 7 14 13 4 16 1 9 4 2 2 5 5 0.008 Untreated Normoxia 0.007 Untreated Hypoxia 0.006 Phe Normoxia 0.005 Phe Hypoxia 0.004 0.003 SNP Normoxia SNP Hypoxia Stroke VolumeStroke (nL) 0.002 0.001 0 N H N H N H N H N H N H 8 15 Days Post Fertilization

Figure 9. Mean  standard error of stroke volume in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to hypoxia (H) or left in normoxia (N) for one hour before examination via microscope.

Cardiac Output

Cardiac output is the volume of blood pumped from the heart per unit time. There were no significant differences on mean cardiac output caused by pharmaceutical treatments (P=0.4832; figure 10). There were insufficient data (figure 11) to perform statistical analyses to determine acute hypoxia effects on cardiac output.

23 1.2 9 5 4 17 15 4 11 12 9 8 5 2

1

0.8 Untreated 0.6 Phe SNP 0.4

0.2

Cardiac OutputCardiac (nlper minute) 0 7 8 14 15 Days Post Fertilization

Figure 10. Mean cardiac output of untreated, Phe treated, and SNP treated Danio rerio.

Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf and the hearts were examined via microscope at 7, 8, 14, and 15 dpf.

24 17 14 13 4 15 0 8 4 2 2 4 5 1.4

1.2 Untreated Normoxia 1 * Untreated Hypoxia 0.8 Phe Normoxia

0.6 Phe Hypoxia SNP Normoxia 0.4 SNP Hypoxia 0.2

Cardiac OutputCardiac (nL per minute) 0 N H N H N H N H N H N H 8 8 15 15 Days post Fertilization

Figure 11. Mean  standard error of cardiac output in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to hypoxia (H) or left in normoxia (N) for one hour before examination via microscope.

Artery Diameter

Arterial diameter was not significantly affected by the pharmaceutical treatments of phenylephrine and sodium nitroprusside (Figure 12). There was no significant difference in arterial diameter between untreated fish versus drug treated fish (P=0.1453).

During acute hypoxia exposure, acute hypoxia caused significant vasodilation in day 15 phenylephrine fish compared to phenylephrine fish that were not exposed to hypoxia

(P=0.0382; figure 13).

25

0.02 46 19 16 53 41 16 37 30 21 21 14 8 0.018 0.016 0.014 0.012 Untreated 0.01 Phe 0.008 SNP

0.006 0.004

Artery Diameter (mm)Artery 0.002 0 7 8 14 15 Days Post Fertilization

Figure 12. Mean  standard error of tail artery diameters in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf and the arteries were examined via microscope at 7, 8, 14, and 15 dpf.

26

53 37 29 16 41 7 21 21 3 8 14 11 0.02 0.018 * 0.016 Untreated Normoxia 0.014 Untreated Hypoxia 0.012 Phe Normoxia 0.01 Phe Hypoxia 0.008 SNP Normoxia 0.006

0.004 SNP Hypoxia Artery Diameter (mm)Artery 0.002 0 N H N H N H N H N H N H 8 8 15 15 Days Post Fertilization

Figure 13. Mean  standard error of tail artery diameters in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to hypoxia (H) or left in normoxia (N) for one hour before examination via microscope. An asterisk denotes a significant difference caused by acute hypoxia exposure compared to normoxia within the respective drug treatment.

Vein Diameter

Veins are part of the cardiovascular system affected by the vasoconstrictory and vasodilatory effects of chronic phenylephrine and chronic sodium nitroprusside treatment. Chronic phenylephrine treatment caused significant vasoconstriction in the vein on day 15 compare to untreated fish (P=0.0491; figure 14). Sodium nitroprusside 27 treatment and acute hypoxia exposure did not have any significant effect on venous diameter.

0.025 46 19 16 53 41 16 37 30 21 21 14 8

0.02

0.015 * Untreated Phe 0.01 SNP

Vein Diameter (mm)Diameter Vein 0.005

0 7 8 14 15 Days Post Fertilization

Figure 14. Mean  standard error of tail vein diameters in Danio rerio. Fish were left untreated (UN) or treated with phenylephrine (Phe) or sodium nitroprusside (SNP) from

1-6 days post fertilization. All drug exposure stopped at 7 dpf and the veins were examined via microscope at 7, 8, 14, and 15 dpf. An asterisk denotes values significantly different (p<0.05) from untreated values within each drug treatment group as determined by Tukey’s test.

28 0.025 53 37 29 16 41 7 21 21 3 8 14 11

0.02 Untreated Normoxia Untreated Hypoxia 0.015 Phe Normoxia Phe Hypoxia 0.01 SNP Normoxia SNP Hypoxia

Vein Diameter (mm)Diameter Vein 0.005

0 8 15 Days post Fertilizaton

Figure 15. Mean  standard error of tail vein diameters in Danio rerio. Fish were left untreated, treated with phenylephrine (Phe), or treated with sodium nitroprusside (SNP) from 1-6 days post fertilization. All drug exposure stopped at 7 dpf. On 8 dpf and 15 dpf half the fish of each treatment was exposed to hypoxia (H) or left in normoxia (N) for one hour. Veins were examined via microscope at 8 and 15 dpf.

29 CHAPTER IV

DISCUSSION

This experiment was designed to determine the effects of chronic exposure to phenylephrine (Phe) or sodium nitroprusside (SNP) on the cardiovascular systems of larval fish. It also was designed to determine how the drug-exposed zebrafish would react physiologically to acute hypoxia. In chronic normoxia, Phe exposure caused a significant increase in heart rate on days 8 and 14 and caused significant vasoconstriction in veins on day 15 (figure 2; figure 14). There were no significant differences in any measured variable in chronically exposed SNP zebrafish in normoxia (figures 2, 4, 6, 8, 10, 12, and

14). Acute hypoxia caused a significant decrease in heart rate in Phe treated fish versus

Phe normoxia exposed fish (figure 3). Acute hypoxia decreased heart rate in SNP fish versus untreated hypoxia fish (figure 3). Phe also caused significant vasoconstriction on day 15 versus untreated fish in the veins. Other tested systems, such as esv, edv, stroke volume, and cardiac output were not significantly affected by chemical treatment or hypoxia exposure.

Chronic Phenylephrine Exposure Effects

Phenylephrine functions by binding to alpha-adrenergic receptors. Once activated, the alpha- pathway produces smooth , or vasoconstriction, in arteries and veins. It is typically used to treat hypotension. Chronic

30 Phe exposure caused several significant changes in developing zebrafish. Chronic Phe exposure caused a significant decrease in heart rate on days 8 and 14, but it was not significantly different on day 15 (figure 2). There was also significant vasoconstriction in the veins on day 15 (figure 14). Chronic Phe did not have an effect on cardiac output, stroke volume, end systolic volume, or end diastolic volume.

Skals et al. (2006) performed a different experiment with a different species

(Synbrachus maroratus) that produced different results than ours, but there are still many similarities between both experiments. In their experiment, Phe exposure made significant decreases in stroke volume and cardiac output, significant vasoconstriction in the central artery and central vein, and heart rate was unaffected. There are similarities that the heart may have been affected the same way in both experiments but used different methods to compensate because they are different species. When vasoconstriction occurs, the heart has work harder to force blood into the artery. In Skals et al. (2006), heart rate did not change but stroke volume significantly decreased. They also had a significant increase in mean circulatory filling pressure. Therefore, blood pressure increased and the heart is compensating by maintaining heart rate but the amount of blood sent from the heart each time is reduced because there is a decreased stroke volume. By comparison, our results showed a significant decrease in heart rate while stroke volume was unaffected. We did not measure pressure to compare, but a decreased heart rate is indicative that there may be a change in pressure. There was no significant difference in artery or vein diameter when the heart rate was significantly reduced, but there was significant vasoconstriction in the veins when the heart rate was not significantly different (figure 2; figure 14). This may have been a compensatory

31 effect. The different responses may be a species-specific development or caused by a difference in acute exposure via injection versus chronic exposure in water of phenylephrine.

Because Phe does not directly cause the heart to beat faster or contract harder, the heart is now forcing the same unit of blood through a smaller artery. The compression of the heart takes longer, slowing down the heart rate (Morelli et al., 2008). On days 8 and

14, after ending chronic Phe treatment, heart rate was significantly decreased. During this same time, there was no significant difference in arterial or venous diameter. It was only on day 15, when heart rate returned to normal that there was significant vasoconstriction in the veins. It is possible that this is a symptom of a chronic effect of Phe treatment. The decrease in heart rate occurs on days 8 and 14, meaning there was possibly still higher blood pressure in the zebrafish larva, possibly from arterial wall modificiation from phenylephrine-induced hypertension (Talmor et al.,1998). By day 15, the zebrafish were further in development and vessel modification may have disappeared as the larva grew

(Gore et al., 2012; Kimmel et al., 1995). This would cause the heart rate to return to normal, but if the zebrafish were physiologically reset to a higher than average blood pressure, this could be compensated by vasoconstriction, as seen in the veins on day 15.

Chronic Sodium Nitroprusside Exposure

During chronic sodium nitroprusside treatment, SNP dissolves and creates nitric oxide. The nitric oxide then activates the guanylate cyclase pathway and causes smooth muscle relaxation. This causes the arteries and veins to vasodilate, decreasing blood pressure, decreasing heart rate, and decreasing stroke volume. In this experiment, SNP

32 did not cause a significant difference in heart rate, cardiac output, end systolic volume, end diastolic volume, stroke volume, arterial diameter, or venous diameter (figures 2, 4,

6, 8, 10, 12, 14).

Previous work by Pelster et al. (2005) examined heart rate and cardiac output in zebrafish during chronic SNP treatment. During early treatment, there was a significant increase in heart rate and a significant increase in cardiac output. Both of these variables showed no significance by days 7 and 8 of data collection, in agreement with our data.

They continued treatment until day 12, when they measured a significant decrease in cardiac output. We assert that because we stopped chronic SNP treatment on day 6, this was the basis for no significant difference in cardiac output on days 14 or 15. Comparing the two studies, it appears early on the heart is significantly affected by chronic SNP treatment, but post-treatment when we examined the fish there were no chronic effects. It is possible that early on the stroke volume is significantly decreased and the heart rate is raised to compensate. The next day, the heart rate returns to normal and stroke volume has increased, causing a significant increase in cardiac output. By day 7, the fish are no longer under chronic SNP treatment and there are no chronic effects from the treatment, leading to no significant differences in treated vs. untreated fish.

In the arteries, previous work by Fritsche et al. (2000) found significant vasodilation in arterial diameter 15 minutes after SNP exposure. We did not find a significant difference in arterial or venous diameter on any day. Comparing the research of the Pelster et al. (2005) and Fritsche et al. (2000), early in development the heart may have been compensating for vasodilation in the veins, but after treatment ended there were no significant differences observed. This means that chronic SNP may have chronic

33 developmental change in the heart, but may not have chronic changes in the arteries or veins.

Acute Hypoxia Exposure

Acute hypoxia caused a significant decrease in the heart rate of chronic Phe and chronic

SNP treated fish (Figure 3). There was also significant vasodilation in the artery of chronic Phe fish exposed to acute hypoxia (Figure 13). Hypoxia did not have an effect on cardiac output, end systolic volume, end diastolic volume, stroke volume, or vein diameter (Figures 5, 7, 9, 11, 15).

Hypoxia affects the vascular system in zebrafish by producing hif-1a. This creates the enzyme nitric oxide synthase, which produces nitric oxide (Hu et al., 2002). This then follows the guanylyate cyclase pathway to cause vasodilation in the arteries and veins.

When exposed to hypoxia, fish treated with chronic Phe had significant vasodilation compared to the unexposed Phe fish. This supports data from Moore et al. (2006) that shows significant vasodilation caused by hypoxia exposure in zebrafish. The acute hypoxia induced vasodilation of chronic Phe treated zebrafish is not significantly different than untreated fish. This was suggested in the second hypothesis that acute hypoxia may antagonize the chronic Phe treatment.

Hypoxia affects the heart by decreasing the available oxygen for oxidative phosphorylation, leading to less ATP production and a reduced heart rate. In this experiment, acute hypoxia exposure caused a significant decrease in the heart rate of Phe treated fish. This supports data that hypoxia will decrease the heart rate (Mendonca et al.,

2010; Bagatto, 2005). Naida et al. (2009) found that the heart rate of zebrafish increased

34 during Phe treatment, therefore this result means that acute hypoxia exposure had a more significant effect than chronic Phe treatment. Phe treatment will have other effects on the rest of the body, particularly an increase in blood pressure and an increase in blood flow, measurements we did not collect but we predict would be affected. Combined with the acute hypoxia vasodilation in the veins of chronic Phe treated fish, there may not be a change in pressure because the blood can flow easier through the expanded veins, but there may be a longer time for circulation of blood because the stroke volume remains unchanged.

Neither acute hypoxia nor chronic SNP treatment caused a significant difference in heart rate when tested seperately, but when the treatments were used in the same fish there was a significant decrease in heart rate. The combination of these two variables creating a significant difference is a little perplexing when compared to previous data

(Figure 3). As stated previously, hypoxia reduces the available oxygen for oxidative phosphorylation, which reduces ATP production, and that causes a decreased heart rate.

Chronic SNP has previously been studied and did not have a significant effect on heart rate in later days of treatment (Pelster et al., 2005). It did, however, create reduced oxygen consumption in the fish during later chronic treatment (Pelster et al., 2005).

Based on these two data points, while chronic SNP may create a reduced oxygen need, the heart still needs an adequate oxygen supply so the reduction in oxygen consumption is probably in other systems.

35 Conclusions

Chronic phenylephrine exposure during early development may have had an effect on blood pressure. Chronic phenylephrine treatment may have caused changes in the arteries and veins that raised the blood pressure, causing heart rate to slow down as the same amount of blood is pushed into a smaller artery (Morelli et al., 2008). If this caused a physiological need for higher blood pressure was normal, it could cause vasoconstriction of the veins to create a higher blood pressure. The presence of chronic changes after phenylephrine treatment ended means that there are chronic developmental differences from the treatment. Chronic sodium nitroprusside exposure did not have a significant effect on development after treatment ended.

Acute hypoxia exposure caused a decrease in heart rate of chronic phenylephrine treated fish and chronic sodium nitroprusside treated fish and significant vasodilation the arteries of chronic phenylephrine treated fish. Acute hypoxia exposure in the heart muscles created a decrease in heart rate despite previous work showing phenylephrine increased heart rate. Acute hypoxia also created significant vasodilation in the arteries of phenylephrine treated fish, so the acute hypoxia exposure had a more significant effect than the chronic phenylephrine treatment.

Final Thoughts and Future Directions

The variation and timing of the significant differences in heart rate and blood vessel diameter was unusual. The significant differences in heart rate did not correspond with the significant differences in vessel dilation. Because there is a temporal delay between heart rate and blood vessel effects, this may be a good area for further

36 examination. For future work I would like to examine the larva earlier in development under pharmaceutical treatment to see how the removal of treatment affects development.

I’d also like to look at blood pressure and blood flow rate under the different treatments to see how they correspond to the data we collected on changes in heart rate and vasoconstriction.

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