CARDIOVASCULAR, METABOLIC, ENDOCRINE AND BEHAVIORAL ASPECTS OF DEVELOPMENT IN POSTNATAL LAMBS IN RELATION TO AGE, SEX,

LAMB NUMBER AND ACUTE FLUOXETINE ADMINISTRATION

by

Tuan-Anh Thi Nguyen

M.D., University Training Center for Health Care Professional, HoChiMinh City, Vietnam, 2001

MSc., The University of British Columbia, 2008

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(REPRODUCTIVE AND DEVELOPMENTAL SCIENCES)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

July 2013

© Tuan-Anh Thi Nguyen, 2013

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Abstract

Human newborns exposed in utero to maternally administered SSRIs such as

fluoxetine (FX) have an increased risk of adverse pregnancy outcomes including poor

neonatal adaptation. This comprises respiratory difficulty, jitteriness, cyanosis when feeding

and persists for several days after birth. Several potential mechanisms underlying these

symptoms have been proposed: 1) acute toxicity to the drugs (i.e. serotonin syndrome), 2)

withdrawal syndrome due to the sudden discontinuation of maternal-fetal placental drug

transfer at birth or 3) an SSRIs-elicited alteration in fetal brain development. However, the

actual mechanism has not been elucidated. In addition, the safety of SSRIs for the treatment

of depression in children and adolescents is uncertain. Thus, we conducted experiments in

which acute i.v FX (1mg/kg) and sterile water were given to FX and control groups in the

lambs at ~ 3,10 days, 1,3,6 and 12 months of age. In another cohort, daily 50mg FX was

given i.v to 5 pregnant ewes via implanted catheters during late gestation (131-132d, term =

147d) for 2 weeks. Plasma FX concentrations in the newborn acute FX group were within the

range measured in human infants at the same age. There was a lack of significant acute FX

effects on cardiovascular-respiratory, behavioral and endocrine functions in ~ 4 day old

lambs. In the lambs exposed to FX prenatally, plasma FX at birth and postnatal day (PND) 2

were low and undetectable on PND 5,10,14. However, prenatal FX-exposed lambs were

hyperactive during PND 4 to14 and their heart rate variability (HRV) was significantly lower

than control lambs on PND 2. Results suggested that SSRI toxicity and withdrawal are

unlikely to be the mechanism underlying poor neonatal adaptation in human exposed to FX

in utero. However, acute FX effects were seen in some, but not all, age groups. No acute FX

effects were observed in the young lambs (10d and 3 months of age). However, hypoactivity,

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transient hypoxemia, hypertension and increased HRV occurred after acute FX

administration in the older lambs (1,6 and 12 months of age). Hypoxemia and hypertension

effects were more profound in males. No changes in HPA axis function were observed.

iv

Preface

I wrote this thesis with direction and input from Dr. Dan Rurak and my supervisory

committee members: Drs. Tim Oberlander, Ken Lim, Wayne Riggs and Anthony Perks.

Surgeries in the ewes and lambs were performed by Dr. Rurak or a clinical veterinarian, Dr.

Bev Chua with assistance from myself and Tim Chow, whose doctoral thesis related to the

determination of the disposition of FX in the postnatal lambs. (Chow, 2013) Daily catheter

flushing and lamb weights were done by myself and Tim. The experiments on the lamb

included in Chapters 3 to 5 were conducted by myself and Tim Chow with occasional

support from our supervisor, Dr. Dan Rurak. I was responsible for collecting heart rate, blood

pressure, heart rate variability data and blood gas samples. Blood gas samples were run by

myself and Dr. Rurak. I was also responsible for collecting plasma samples for ACTH and

cortisol. Tim Chow was responsible for collecting plasma samples for FX measurement and

urine samples as well as developing and running the LC/MS/MS assay. (Chow et al, 2011)

Thus the plasma FX concentration versus time curve in figure 4.1 is from Tim’s thesis.

However, although Tim analyzed the plasma FX concentrations in Chapter 6, these data are

not part of his PhD thesis, I did all the remaining data analysis from actiwatch, Digital Video

Recording (DVR), power lab, ECG, blood gas, ACTH and cortisol assay.

All experiments, sample collection and data analysis in the ewes and lambs included

in Chapter 6 were done by myself. The catheter implantation procedure in the pregnant ewes

was performed by Dr. Bev Chua with assistance from myself, Dr. Rurak and the staff in the

Centre of Comparative Medicine (CCM). FX measurement in this chapter was also run by

Tim Chow.

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Manuscripts will be prepared for future publication based on results from Chapters 3-

6. Manuscripts will include relevant sections found in Chapters 1-7 including introduction,

methods, results and discussions.

This thesis was conducted under animal care approval from the University of British

Columbia Animal Research Ethics (Certificate number: A07-0302)

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Table of Contents

Abstract .................................................................................................................................... ii 

Preface ..................................................................................................................................... iv 

Table of Contents ................................................................................................................... vi 

List of Tables .......................................................................................................................... ix 

List of Figures .......................................................................................................................... x 

List of Symbols and Abbreviations .................................................................................... xvi 

Acknowledgements ............................................................................................................ xviii 

Dedication ............................................................................................................................. xix 

1. Introduction ......................................................................................................................... 1 1.1  Postnatal Lamb Development: .............................................................................................. 1 

1.1.1  Cardiovascular Function: .................................................................................................. 1 1.1.2  Homeostasis And Metabolism: ......................................................................................... 2 1.1.3  Behavior: Maternal-Infant Bonding: ................................................................................ 4 1.1.4  Hypothalamic-Pituitary-Adrenal (HPA) Axis Function: .................................................. 6 1.1.5  Renal Function: ................................................................................................................. 7 

1.2  Depression: Symptoms And Diagnosis:................................................................................ 8 1.3  Pathophysiology Of Depression: ........................................................................................ 10 1.4  Depression In Pregnancy, Postpartum And In Children: .................................................... 16 

1.4.1  Incidence: ........................................................................................................................ 16 1.4.2  Risk Factors Of Depression: ........................................................................................... 17 1.4.3  Effects Of Maternal Depression On Fetal Neurobehavioral Development: ................... 19 1.4.4  Treatment Of Depression: ............................................................................................... 20 

1.5  Selective Serotonin Reuptake Inhibitors (SSRIs): .............................................................. 21 1.5.1  Clinical Uses: .................................................................................................................. 21 1.5.2  Mechanism Of Action: ................................................................................................... 21 1.5.3  Pharmacokinetics: ........................................................................................................... 24 1.5.4  Side Effects: .................................................................................................................... 26 

1.6  Fluoxetine (FX): .................................................................................................................. 27 1.6.1  Pharmacokinetics: ........................................................................................................... 28 

1.6.1.1  Absorption: ............................................................................................................ 28 1.6.1.2  Distribution: ........................................................................................................... 28 1.6.1.3  Metabolism: ........................................................................................................... 28 1.6.1.4  Elimination: ............................................................................................................ 29 

1.7  FX Effects On Newborns And Infants: ............................................................................... 29 1.7.1  FX Effects On Cardiovascular Functions: ...................................................................... 29 1.7.2  FX Effects On The Homeostatic And Respiratory Functions: ....................................... 32 1.7.3  FX Effects On Birth Outcomes: ..................................................................................... 32 1.7.4  FX Effects On Neonatal And Child Behaviors: .............................................................. 34 1.7.5  FX Effects On The Hypothalamic-Pituitary-Adrenal (HPA) Axis: ................................ 37 

1.8  Rationale: ............................................................................................................................ 39 1.9  Objectives: .......................................................................................................................... 41 1.10  Hypotheses: ......................................................................................................................... 42 

2. Materials and Methods ..................................................................................................... 43 

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2.1.1  Animals: .......................................................................................................................... 43 2.1.2  Surgical Preparation: ...................................................................................................... 44 2.1.3  Experimental Protocol: ................................................................................................... 46 2.1.4  Sample Collection:.......................................................................................................... 48 2.1.5  Digital Video Recording And Behavioral Evaluation: ................................................... 49 2.1.6  Actiwatch Rest/Activity Monitoring: ............................................................................. 50 2.1.7  Powerlab Data Acquisition System: ............................................................................... 51 2.1.8  Arterial Blood Gases Analysis: ...................................................................................... 52 2.1.9  Adrenocorticotropin Hormone Assay: ............................................................................ 52 2.1.10  Cortisol Assay: ........................................................................................................... 53 2.1.11  FX and NFX assay: .................................................................................................... 53 2.1.12  Statistical Analysis: .................................................................................................... 54 

3. Postnatal Lamb Development and Sex Differences ....................................................... 55 3.1  Introduction: ........................................................................................................................ 55 3.2  Methods: ............................................................................................................................. 57 

3.2.1  Experimental Protocol: ................................................................................................... 57 3.2.2  Statistical Analysis: ........................................................................................................ 57 

3.3  Results: ................................................................................................................................ 58 3.3.1  The Development Of Physiological Variables And Sex Differences In Development From The Newborn Period Up To 1 Year Of Age: ..................................................................... 58 3.3.2  Neonatal Behavioral Development: ................................................................................ 72 3.3.3  The Development Of Circadian And Ultradian Rhythms. The Ultradian 1 h Rhythm Is Associated With Suckling: .......................................................................................................... 74 3.3.4  Weaning And Changes In Physiological Variables: ....................................................... 80 

3.4  Discussion: .......................................................................................................................... 83 

4. Acute Fluoxetine Effects In The Newborn...................................................................... 99 4.1  Introduction: ........................................................................................................................ 99 4.2  Methods: ........................................................................................................................... 102 

4.2.1  Experimental Protocol: ................................................................................................. 102 4.2.2  Statistical Analysis: ...................................................................................................... 102 

4.3  Results: .............................................................................................................................. 103 4.3.1  Plasma FX Concentration And Weight Gain: ............................................................... 103 4.3.2  Rest Activity Cycles: .................................................................................................... 105 4.3.3  Cardiovascular Variables (Heart Rate, Arterial Pressure, Heart Rate Variability): ...... 105 4.3.4  Arterial Blood Gases: ................................................................................................... 109 4.3.5  Plasma Cortisol Concentration: .................................................................................... 112 

4.4  Discussion: ........................................................................................................................ 113 4.4.1  Acute FX Effects On Cardiovascular –Respiratory Functions: .................................... 115 4.4.2  Acute FX Effects On HPA Axis Functions: ................................................................. 118 4.4.3  Underlying Mechanisms Of Poor Neonatal Adaptation: .............................................. 119 

5. Acute Fluoxetine Effects In The Postnatal Lambs (From 10 Days To 1 Year Of Age)............................................................................................................................................... 123 

5.1  Introduction: ...................................................................................................................... 123 5.2  Methods: ........................................................................................................................... 125 5.3  Results: .............................................................................................................................. 127 

5.3.1  Acute FX Effects On Cardiovascular-Respiratory Functions: ...................................... 127 5.3.2  Acute FX Effects On Homeostatic And Respiratory Functions: .................................. 131 5.3.3  Acute FX Effects On Behavior: .................................................................................... 141 

5.3.3.1  Behavioral Activities In The Lambs At 1 Month Of Age: ................................... 141 

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5.3.3.2  Behavioral Activities In The Lambs At 6 Months Of Age: ................................. 148 5.3.3.3  Acute FX Effects On Cortisol Level: ................................................................... 153 

5.4  Discussion: ........................................................................................................................ 156 

6. Postnatal Outcomes in Lambs Exposed Antenatally to FX ........................................ 167 6.1  Introduction: ...................................................................................................................... 167 6.2  Methods: ........................................................................................................................... 169 

6.2.1  Animal And Catheter Implantation: ............................................................................. 169 6.2.2  Experimental Protocol: ................................................................................................. 170 6.2.3  Digital Video Recording And Behavioral Evaluation: ................................................. 171 6.2.4  Actiwatch Rest/Activity Monitoring: ........................................................................... 172 6.2.5  Powerlab Data Acquisition System: ............................................................................. 172 6.2.6  Pulse Oximeter: Tissue SpO2 And Heart Rate:............................................................. 172 6.2.7  FX And NFX Concentration: ........................................................................................ 173 6.2.8  Statistical Analysis: ...................................................................................................... 173 

6.3  Results: .............................................................................................................................. 174 6.4  Discussion: ........................................................................................................................ 187 

7. Summary and Conclusions............................................................................................. 194 

REFERENCES .................................................................................................................... 215 

Appendices ........................................................................................................................... 252 Appendix 1: Actiwatch Variables Data In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age: ................................................................................................................................................ 252 Appendix 2: Heart Rate, Arterial Pressure And HRV In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection: ............................................................................................... 255 Appendix 3: Arterial Blood Gases And Body Temperature In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection: .................................................................................. 258 Appendix 4: Glucose, Lactate Concentration And Electrolite In The Lambs At~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection: .................................................................................. 261 

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List of Tables

Table 1.1: DSM-IV-TR criteria for major depressive episode ............................................... 10 

Table 2.1: Time points at which blood samples were collected for arterial blood gases, FX, ACTH and cortisol measurement. SW = sterile water. .......................................................... 48 

Table 3.1: The time window of weaning in lambs was defined as when lambs started to first consume solid food to when 1h rhythm due to suckling disappeared .................................... 82 

Table 3.2: Break points of cardiovascular, metabolic, endocrine and renal variables. ........... 82 

Table 6.1: Gestational age and birth weight in the control and antenatally FX-exposed lambs............................................................................................................................................... 175 

Table 6.2: Mean ± SEM of HRV values for the first 30 mins of the experiment in the lambs on day 2 and the changes from day 5, 10, 14 compared with the values on day 2 ............... 186 

Table 7.1: Outcomes of chronic antenatal and acute postnatal FX exposure in fetal and postnatal lambs ...................................................................................................................... 206 

Table 7.2: Outcomes of antenatal SSRI exposure in human (exposure for most or all of pregnancy) ............................................................................................................................. 207 

Table 7.3: Postnatal outcomes of antenatal and postnatal SSRI exposure in rodents ........... 212 

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List of Figures

FIG. 1.1: Biosynthesis and metabolism of serotonin. ............................................................. 15 

FIG. 1.2: Schematic diagram of monoaminergic synapse and steps involved in synaptic transmission: Monoamine neurotransmitters (1) are synthesized within neurons from common amino acid precursors, which are Tryptophan and 5-Hydroxytryptophan (5-HT) in the case of serotonin, (2) are taken up into synaptic vesicles via a vesicular monoamine transporter, (3) are released into the synaptic cleft upon stimulation, (4) interact with postsynaptic receptors to alter the excitability of postsynaptic cells, (5) interact with presynaptic autoreceptors located on the nerve terminal to suppress further release. (6) Released monoamines may also be taken back up from the synaptic cleft into the nerve terminal by plasma membrane transporter proteins. (7) Once taken up, monoamine may be subject to enzymatic degradation or may be protected from degradation by uptake into vesicles. MAO: monoamine oxidase ...................................................................................... 22 

FIG. 3.1: Postnatal lamb arterial pressure (A) and heart rate (B) in all (open bar), male (black bar) and female (gray bar) lambs at 3 days (n = 14), 10 days (n = 18), 1 month (n = 15), 3 months (n = 17), 6 months (n = 11) and 1 year (n = 10) of age. ............................................. 60 

FIG. 3.2: Time domain of heart rate variability: SDNN (standard deviation of NN interval) (A), SD Delta NN (Standard Deviation of the differences between adjacent NN intervals) (B), RMSSD (Square root of the mean of the squared differences between adjacent NN intervals) (C) in lambs at 3 days (n = 11), 10 days (n = 18), 1 month (n = 15), 3 months (n = 17), 6 months ( n = 11) and 1 year ( n = 10) of age. ............................................................... 61 

FIG. 3.3: Frequency domain of heart rate variability: LF (Low Frequency) power (A), HF (High Frequency) power (B), LF/HF ratio (Low Frequency/High Frequency Ratio) (C) in lambs at 3 days (n = 11, male n = 4, female n = 7 ); 10 days (n= 18, male n = 9, female n = 9), 1 month (n= 15, male n = 8, female n = 7); 3 months (n= 17, male n = 9, female n = 8), 6 months (n = 11, male n = 6, female n = 5), 1 year (n = 10, male n = 6, female n = 4) of age. 62 

FIG. 3.4: The relationship between SDNN (msec) and heart rate (bpm) in the lambs from 3 day to 1 year of age ................................................................................................................. 63 

FIG. 3.5: Mean pH value in total (open bar), male (black bar) and female (gray bar) lambs at 3 days (n = 13, male n = 6, female n = 7 ); 10 days (n= 17, male n = 8, female n = 9), 1 month (n= 15, male n = 8, female n = 7); 3 months (n= 17, male n = 9, female n = 8), 6 months (n = 10, male n = 5, female n = 5), 1 year (n = 10, male n = 6, female n = 4) of age. 65 

FIG. 3.6: Mean pCO2 (A), pO2 (B) and O2 sat (C) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. ....... 66 

FIG. 3.7: Mean HCO3 (A), Base Excess (B) and Hemoglobin (C) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. .......................................................................................................................................... 67 

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FIG. 3.8: Mean Glucose (A) and Lactate (B) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. ................. 68 

FIG. 3.9: Mean urine flow rate in lambs at 3 days (n = 10); 10 days (n= 10), 1 month (n= 9); 3 months (n= 12); 6 months (n = 7) and 1 year (n = 10) of age. ............................................. 69 

FIG. 3.10: Mean cortisol concentration in fetal lambs from preinfusion to infusion day 2 and from infusion day 5 to 8 adapted from Morrison et al, 2004 and in postnatal lambs prior surgery (n = 11), at 3 days (n = 14); 10 days (n= 12), 1 month (n= 12); 3 months (n= 12); 6 months (n = 8) and 1 year (n = 8) of age. ............................................................................... 70 

FIG. 3.11: Mean ACTH concentration in fetal lambs from preinfusion to infusion day 2 and from infusion day 5 to 8 adapted from Morrison et al, 2004 and in postnatal lambs prior surgery (n = 11), at 3 days (n = 2); 10 days (n= 2), 1 month (n= 2); 3 months (n= 14); 6 months (n = 3) and 1 year (n = 4) of age. ............................................................................... 71 

FIG.3.12: Cortisol concentration during the day in the lambs at 10 days, 1 month and 1 year of age. ...................................................................................................................................... 73 

FIG. 3.13: Weight gain (A) and total suckling time (B) in singleton (n = 6), twin (n =14 for weight gain and n = 10 for total suckling time) and triplets (n = 3) in lambs on postnatal day 2-3 (N = 22). ........................................................................................................................... 76 

FIG. 3.14: Plots of daily total activity score (a), activity bouts/day (b), mean score in active period (c), mean episode duration (d), % time moving (e) and % time immobile (f) against postnatal age in lambs. (n=21) ................................................................................................ 77 

FIG. 3.15: (A) The average development of 24, 8, 6 and 1-2 hour activity-rest rhythms. The lines with diamond, square, triangle and x symbols indicate the 24h circadian, the 8 hour, the 6 hour and 1 hour ultradian rhythms, respectively. Levels of rhythm dominance of 1 to 4 indicate first to fourth most dominant rhythms. A level of rhythm dominance of greater than 4 indicates that this rhythm is not one of the top four rhythms (n=22). (B) The daily mean total activity score. (n= 21) ..................................................................................................... 78 

FIG. 3.16: (A). Relationship between the day that the 1h rhythm disappears and the day of the lowest total activity (n = 21). (B) Relationship between the day that the 6h rhythm disappears and the first day lamb eating hay (n = 21) ............................................................ 79 

FIG. 3.17: Relationship between suckling frequency on postnatal day 2-3 from DVR observation and calculated from the actiwatch FFT (n = 21) ................................................. 80 

FIG. 4.1: (A) Plasma FX concentration verus time curve in the 3 day-old lambs (closed symbols and solid line) and in 13 individual human infants (open symbols) sampled at postnatal day 2 following in utero exposure to the drug. Human data was taken from the dissertation of Dr. John Kim, 2000 and the lambs data was taken from the dissertation of Dr. Tim Chow, 2013. (B) Weight gain on ~ postnatal day 4 among the normal term lambs (n = 11, open bar), control group (n = 9, black bar), FX group (n = 12, gray bar) and post FX day 1 (n = 12, light gray bar) ....................................................................................................... 104 

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FIG. 4.2: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in the normal ~ 4 day-old term lambs (n = 10), control = on day of control experiment (n=6), and FX lambs on the day of FX experiment (n = 11) followed up to post FX day 1 (n = 11), post FX day 2 (n = 11) and post FX day 3 (n = 11). No significant changes were found between activities after FX administration and sterile water injection or normal day without experiments. ................... 106 

FIG. 4.3: Arterial pressure and Heart rate (at 1 min intervals for the first 5 min after dosing and at 5 minute interval for the whole 2.5h of experiment) in the ~ 4 days old lamb with sterile water injection (control group) and FX injection (FX group). ................................... 107 

FIG. 4.4: Time domain of HRV (SDNN, SD Delta NN, RMSSD) and frequency domain (LF power, HF power, LF/HF ratio) at 30 min intervals in the newborn lambs at ~ 4 days old in the control group (open diamond and dotted line) and FX group (close circle and solid line)............................................................................................................................................... 108 

FIG 4.5: The relationship between changes in heart rate at 2 mins following FX (delta HR) and changes in time domain of HRV (SDNN, SD Delta NN, RMSSD) (i.e Delta SDNN, Delta SD Delta NN, Delta RMSSD) at 1 min following FX in the lambs at ~ 4 days of age................................................................................................................................................ 110 

FIG. 4.6: Arterial blood gases (pH, O2 saturation, pO2, pCO2, HCO3, Base Excess) in the newborn lambs (~ 4 days old) in the control group (open diamond and dotted line, n = 9) and FX group (close circle and solid line, n = 11) ...................................................................... 111 

FIG. 4.7: Glucose, Lactate and Hb concentration in the newborn lambs (~ 4 days old) in the control group (open diamond and dotted line, n = 9) and FX group (close circle and solid line, n = 11) ........................................................................................................................... 112 

FIG. 4.8: Plasma cortisol concentration in the ~ 4 days old lambs in the control group with sterile water injection (n = 8, open bar) and the FX group with FX injection (n = 5, close bar) at 30 and 15 minutes before injection and 0.5, 1, 2, 6, 12 hours after injection ................... 113 

FIG 5.1: Arterial pressure and heart rate changes in 1 year old lambs with acute FX administration ....................................................................................................................... 128 

FIG 5.2: HRV (SDNN, SD Delta NN, RMSSD, LF power, HF power, LF/HF ratio) changes with acute FX administration in 1 year old lambs. ............................................................... 130 

FIG 5.3: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 1 month of age. ....................................................................................................... 132 

FIG 5.4: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 6 months of age. ..................................................................................................... 133 

FIG 5.5: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 1 year of age. .......................................................................................................... 136 

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FIG 5.6: (A) The relationship between changes in pO2 at 5 mins and changes in arterial pressure (AP) at 2 mins following FX injection in all lambs at 1 year of age (square symbol, solid line, n = 8); (B) The relationship between changes in pO2 at 5 mins and changes in AP at 2 mins following FX injection in male (square symbols, dotted line, n = 4) and female (circle symbols, solid line, n = 4) at 1 year of age and (C) The relationship between changes in O2 saturation at 5 mins and changes in AP at 2 mins following FX injection in male (square symbols, dotted line, n = 4) and female (circle symbols, solid line, n = 4) at 1 year of age. ........................................................................................................................................ 137 

FIG 5.7: (A) The relationship between changes in pO2 at 5 mins and log FX concentration at 5mins following FX injection in all lambs at 1 year of age (square symbol, solid line, n = 8) (B) The relationship between changes in pO2 at 5 mins and log FX concentration at 5mins following FX injection in male (triangle symbols, dotted line, n= 4) and female (circle symbols, solid line, n = 4) lambs at 1 year of age; and (C) The relationship between changes in O2 saturation at 5 min and log FX concentration at 5mins following FX injection in male (triangle symbols, dotted line, n= 4) and female (circle symbol, solid line, n = 4) lambs at 1 year of age. ............................................................................................................................ 138 

FIG 5.8: PCO2 , base excess (BE), HCO3 concentrations with sterile water and FX administration in male and female lambs at 1 year of age. ................................................... 139 

FIG 5.9: Glucose, lactate, Hb (Hemoglobulin) concentrations with sterile water and FX administration in male and female lambs at 1 year of age. ................................................... 140 

FIG. 5.10: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in 1 month-old lambs with the average of the 4 normal days in the pen before the experiments (n = 12, open bar), on control experiment day (n=9, black bar), and on FX experiment day (n = 12, dark gray), post FX day 1 (n = 11, light gray), post FX day 2 (n = 11) and post FX day 3 (n = 11). ......................... 144 

FIG. 5.11: (A) Percentage of time lambs spent for each activity from DVR observation and (B) the intensity of each activity when matching DVR observation with actiwatch score data in lambs at 1 month of age. Normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). ................................................................................................. 145 

FIG. 5.12: (A) Percentage of time lambs spent for standing activity from DVR observation and (B) the intensity of standing quiet and standing moving when matching DVR observation with actiwatch score data in lambs at 1 month of age. Normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar)........................................................................... 146 

FIG 5.13: Weight gain (mean ± SE) in the lambs during their normal days, control experiment with distilled water injection, FX experiment with FX injection and 3 continuous days after the FX experiment day around 1 month of age. ................................................... 147 

FIG. 5.14: Intensity of standing and playing activity from DVR observation in the 1 month-old male and female lambs on normal day (day lambs in the pen without any experiments,

xiv

gray bar, n = 3), on Control experiment day (n=6, open bar) and FX experiment day (n=6, black bar). ............................................................................................................................. 147 

FIG. 5.15: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in 6 month-old lambs with average of 4 normal days in the pen before the experiments (n = 8, open bar), on control experiment day (n=8, black bar), and on FX experiment day (n = 8, dark gray bar), post FX day 1 (n = 8), post FX day 2 (n = 8) and post FX day 3 (n = 8). ................................................................. 150 

FIG. 5.16: (A) Percentage of time lambs spent for each activity from DVR observation and (B) the intensity of each activity when matching DVR observation with actiwatch score data in lambs at 6 month of age. The normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). ................................................................................................. 151 

FIG. 5.17: (A) Percentage of time lambs spent on standing (quiet and moving) activity from DVR observation and (B) the intensity of standing quiet and standing moving when matching DVR observation with actiwatch score data in lambs at 6 month of age. The normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). ....................... 152 

FIG 5.18: Weight gain (mean ± SE) in the lambs during their normal days, control experiment with distilled water injection, FX experiment with FX injection and 2 continuous days after the FX experiment day at around 6 months of age. ............................................. 153 

FIG. 5.19: Cortisol and ACTH concentration in the lambs at 3 months of age at 15, 30 minutes before and 0.5, 1, 2, 6 and 12 h after sterile water injection (control group) and FX injection ( FX group) ............................................................................................................ 154 

FIG. 5.20: Cortisol and ACTH concentration in the lambs at 1 year of age at 15, 30 minutes before and 0.5, 1, 2, 6 and 12 h after sterile water injection (control group) and FX injection (FX group) ............................................................................................................................ 155 

FIG. 6.1: (A) Daily weight in the control (n=7) and prenatal FX-exposed lambs (n=7) from postnatal day 1 (day of birth) to postnatal day 14. ................................................................ 175 

FIG.6.2: FX, (R)-FX, (S)-FX and NFX, (R-)NFX, (S)-NFX concentrations in the prenatally FX-exposed ewes (n= 5) and lambs (n=7). Samples were taken at birth in both ewes and lambs and on postnatal day 2,5,10 and 14 only in lambs. .................................................... 177 

FIG 6.3: The relationship between the lamb:ewe plasma FX concentration ratio and the time from the last FX dose (hours) in the ewe. ............................................................................. 178 

FIG. 6.4: Daily (A) tissue spO2 and (B) heart rate measured by pulse oximeter in the control (n=2) and prenatal FX-exposed lambs (n =7) from postnatal day 1 (day of birth) to postnatal day 14. ................................................................................................................................... 178 

xv

FIG. 6.5: Neonatal behavior (first time to stand, walk, suck immediately after birth) in the control lambs (n = 37) and prenatal FX-exposed lambs (n =7). ........................................... 181 

FIG. 6.6: Daily activity variables (active bouts per days, total activity score, mean score in active, episode duration, % time mobile, % time immobile) obtained from the actiwatches in the control (opened, diamond symbols) and prenatal FX-exposed lambs (closed, circle symbols) from postnatal day 2 (the first full day) to postnatal day 14. ................................ 183 

FIG. 6.7: Daily sleep, suckling and non-suckling activity bouts obtained from DVR observations in the control (open, diamond symbols) and prenatal FX-exposed lambs (closed, circle symbols) from postnatal day 2 (the first full day) to postnatal day 10. ...................... 184 

FIG. 6.8: HRV (time domain: SDNN, SD Delta NN, RMSSD; frequency domain: total power, LF power, HF power, LF/HF) on postnatal day 2 in control group (diamond symbol, dotted line, n = 8) and prenatal FX-exposed lambs (circle symbol, solid line, n = 7). ......... 185 

xvi

List of Symbols and Abbreviations 5-HIAA 5-hydroxyindoleacetic acid 5-HTP 5-hydroxy-tryptophan 5-HT 5-hydroxy-tryptamine/ Serotonin ACTH Adrenocorticotrophin ANS Autonomic Nervous System AUC Area Under the Curve BE Base Excess CBT Cognitive-Behavioral Therapy CCM Centre of Comparative Medicine CRH Corticotropin-Releasing Hormone CYP Cytochrome P450 DVR Digital Video Recording FDA Food and Drug Administration FFA Free Fatty Acid FFT Fast Fourier Transformation FX Fluoxetine GA Gestational Age GFR Glomerular Filtration Rate GH Growth Hormone GLM General Linear Model HF power High Frequency power HPA Hypothalamic-Pituitary-Adrenal HRV Heart Rate Variability IGF-1 Insulin-like Growth Factor-1 LF power Low Frequency power LF/HF ratio Low Frequency/High Frequency power ratio MAOI Monoamine Oxidase Inhibitors MCA Middle Cerebral Artery MDD Major Depressive Disorder msec Millisecond NaSSA Noradrenergic and Specific Serotonergic Antidepressant NDRI Norepinephrine/Dopamine Reuptake Inhibitor NFX Norfluoxetine nu normalized unit OCD Obsessive Compulsive Disorder PMSG Pregnant Mare Serum Gonadotropin PND Postnatal Day PPHN Persistent Pulmonary Hypertension RCT Randomized Controlled Trial REM Rapid Eye Movement RMSSD Square root of the mean of the squared differences between adjacent

NN intervals. SARI Serotonin Antagonist Reuptake Inhibitor

xvii

SD Delta NN Standard Deviation of the differences between adjacent NN intervals SDNN Standard Deviation of the NN intervals SEM Standard Error of Mean SNRIs Serotonin-Norepinephrine Reuptake Inhibitors sO2 Oxygen saturation SSRIs Selective Serotonin Reuptake Inhibitors TCA Tricyclic Antidepressant/Tricyclic Acids VFA Volatile Fatty Acid WHO World Health Organization

xviii

Acknowledgements

I would like to express my deepest gratitude to my supervisor and mentor, Dr.Dan

Rurak for his guidance, dedication, and support over the course of my PhD study. I would

also like to thank my supervisory committee: Drs.Wayne Riggs, Tim Oberlander, Ken Lim

and Anthony Perks for their encouragement and valuable advice for my project; and my

special thanks to Tim Chow for his invaluable assistance in the lab experiments. I would also

appreciate Dr. Bev Chua for her friendship and her dedicated care toward the sheep. Thanks

to all the staffs in the Child and Family Research Institute (CFRI) and Centre of Comparative

Medicine (CCM): Catherine, Edwards, Leika, Claire, Gordon and Kelly for their animal care

knowledge. I would also like to thank Roshni Nair for her continuous support and

compassion towards me during my entire graduate program. Thanks to my friends in the

Reproductive and Developmental Sciences program for their positive engagement.

My warmest thanks to Rev. Joshep Nguyen; all seniors from the St. Andrew’ parish

as well as my friends for their help and making my life more functional outside of the school.

Most of all, I would like to sincerely thank my brother, Sean Nguyen, whom I am

most grateful for his sacrifice helping out with the family needs allowing me to focus on my

study. Above all I want to thank my mom and late dad for teaching me the value of

education. Without your unconditional and steadfast love, I cannot be who I am today.

A final thanks to all the funding sources for the project and my studentship (CIHR,

IWRH, CFRI).

xix

Dedication

To the memories of my father

To my beloved mom and brother

1

1. Introduction

1.1 Postnatal Lamb Development:

1.1.1 Cardiovascular Function:

Birth is an event which involves remarkable changes in the environment surrounding

the fetus. Moving from a comfortable warm environment, filled with fluid and maternal soft

tissue as a cushion, and readily available nutrients and oxygen supply via the placenta in

utero to an extra utero life, the newborn for the first time is exposed to cold challenge,

gravity, air, and hard surfaces. In order for a successful transition to be accomplished, it is

required that the newborn undergo a series of physiological changes (such as air breathing,

shift in cardiovascular patterns, thermoregulation, alteration of oxygen transport (Lister et al.,

1984) and behavioral adaptations (e.g: becoming aroused and aware of the surrounding

environment, being responsive and interactive in order to start establishing bonding with the

mother, and initiating suckling (Mellor & Gregory, 2003; Mellor, 1988).

After birth, the high relative cardiac output observed in the fetus is still maintained in

the newborn lambs up to 1 month of age (Minoura & Gilbert, 1987). One of the reasons

explained for the high cardiac output in the newborn lambs is that more O2 needs to be

transported to the tissues since O2 extraction is limited by the avid binding of O2 to

hemoglobin (Lister et al, 1979). As there is an increasing oxygen consumption in the

newborn lamb in response to cold exposure after birth (Alexander & Williams, 1968; Sidi et

al., 1983), it is vital for them to maintain a high cardiac output. The subsequent decline in

heart rate over the first 2 months of life is proportionate to that of cardiac output to keep the

stroke volume relatively constant in relation to the body weight (Lister et al., 1979). In

2

lambs, cardiac sympathetic innervations and parasympathetic influences on the heart are

incomplete at birth. However, the sympathetic nervous system appears to be predominant in

the lamb less than 1 week of age whereas parasympathetic becomes dominant in lambs older

than 3 months. It is believed that the sympathetic activity and circulating catecholamines help

to maintain such high cardiac output in 1-3 day old lambs (Minoura & Gilbert, 1987). Siimes

et al have reported that the beat-to-beat heart rate variability (HRV) increased in lambs after

3 weeks of age (Siimes et al., 1984). This increasing trend of HRV with advancing age is

similar to that observed in human infants (Harper et al., 1976; Wheeler et al., 1979). In

addition, HRV varies in relation to sleep state. It is highest during waking and active sleep

and lowest during quiet sleep (Siimes et al., 1984)

1.1.2 Homeostasis And Metabolism:

Since nutrients are ingested orally after birth, before the newborns are able to nurse

from their mother and before maternal milk production increases, they have to use their own

energy reserves to produce heat against the cold stress. In newborn lambs, both shivering

(muscle glycogenolysis) and non-shivering metabolism (or brown adipose tissue

thermogenesis) are equally important in cold-induced thermogenesis. In brown adipose tissue

thermogenesis, the brown adipocyte was stimulated by noradrenaline via ß-adrenergic

receptors. This gives rise to a series of actions with a final release of free fatty acid (FFA)

within the cell. Even though these FFA might be released to the circulation, a significant

amount of these FFA remain in the cell and is transferred to the mitochondria where they are

oxidized to produce heat via the electron transport change and action of brown adipose

specific uncoupling protein (Symonds et al., 2012). With advancing age, the non-shivering

contribution declines rapidly while shivering metabolism increases (Alexander & Williams,

3

1968; Alexander et al., 1968, Symonds et al., 2012). However, lambs exposed to cold

showed a much greater increase in free fatty acid concentration in those older than 20 days

than those at 1-3 days old (Alexander et al., 1968). As the authors explained, this is because

the proportion of brown adipose tissue, which can re-utilize fatty acids from triglyceride

lipolysis, decreases while white adipose tissue increases over age, thus during cold exposure

in the older lambs, the rate of lipolysis exceeds the rate of FFA recycling within the adipose

cell (Alexander et al., 1968).

In the transition from prenatal life, in which nutrient supply and growth are

increasingly constrained by the placenta, to postnatal life with major changes in the quantity

and composition of nutrient supply, there is a requirement for metabolic adaptations. This

includes colonization of the digestive system, which begins soon after birth (Morrison et al.,

2009). The composition of nutrients in utero is primarily glucose and amino acids whereas a

diet after birth may contain a high level of fat and less carbohydrate (Girard et al., 1997;

Greenwood et al., 2002). In sheep, with the dietary switch from milk-fed to solid feed at

weaning, the morphology and metabolic aspects of the ruminant digestive system also

change accordingly. The rumen increases in size, the intraruminal papillae increase in length

and the epithelium lining the rumen becomes keratinized (Lake et al., 1982; Lane, Baldwin,

& Jesse, 2000; Lavker, Chalupa, & Opliger, 1969). In the presence of solid feed, ruminal

microorganisms produce volatile fatty acids (VFA) via fermentation of the feed. VFA

exposure appears to be necessary to stimulate rumen metabolic development. Examining

rumen development in lambs with and without solid feed in their diet, Lane et al have

concluded that the presence of solid food is needed for rumen metabolic maturation (Lane et

al., 2000).

4

In a study of blood composition in normal and growth retarded lambs over the first 24

hour after birth, Mellor & Pearson (1977) found that plasma glucose and IgG increase a few

hours after birth, which is most likely attributed to the absorption of colostrum from the gut.

In contrast, lactate concentration decreases sharply after birth. Plasma fructose concentration

was decreased progressively in late gestation fetal lambs till birth and had disappeared from

the lamb’s plasma by the first day of birth (Comline & Silver, 1970).

A recent study in our lab has found that arterial Po2, pH, BE, So2 decrease and Pco2

and hemoglobin concentration increase in the fetal lambs with advancing gestational age

(Rurak & Bessette, 2012). However, to date there is limited information on the changes in

arterial blood gas values of newborn and postnatal lambs in response to cardiovascular-

respiratory development as well as other physiological processes.

1.1.3 Behavior: Maternal-Infant Bonding:

Different from rodents, which are altricial at birth, lambs are precocial and born at an

advanced stage of physical development. In addition, all sense organs (tactile, olfactory,

auditory and visual) of the lamb are fully-developed and ready to function from birth (Fraser

& Broom, 1997). This allows mutual recognition between ewes and lambs, which happens

very early following birth, and in which visual appearance of the lamb appears to be an

important cue (Walser & Alexander, 1980). After the first two hours of parturition, cross

fostering lambs is no longer possible, even when the alien lambs were masked by their foster

mother’s amniotic fluid (Walser & Alexander, 1980). Indeed, maternal-infant bonding is first

established immediately after delivery by a coordinated expression of behaviors between

both the ewe and lambs. Together with the ewe’s behavior at birth such as licking or

5

grooming, rumbles (frequent low-pitched bleats made by the ewes as she licks the lamb dry)

and udder acceptance, it is also vital for the lamb to successfully accomplish a sequence of

behaviors directed toward standing, finding the udder and suckling during this immediate

postpartum period (Fraser & Broom, 1997). These behaviors from the ewe serve not only for

the formation of a strong maternal-infant attachment with her own lamb but also as an

expression of nurturing behavior to facilitate the successful transition from the pre to

postnatal life for the lambs (Dwyer, 2008a). Normally, the ewe licks to dry the lamb, to clear

placental membranes from the lamb’s nose and mouth, to stimulate activity and respiration

and to form an olfactory memory for the lamb. In addition, a rumbling sound made by the

ewe when she licks the lamb may help to reduce stress in the lamb (Dwyer et al., 1998) and

form the auditory memory for later maternal recognition (Walser & Alexander, 1980). These

sound cues also strongly influence the lamb’s behavior. It helps the lambs to orientate to the

ewe and keep them near to the ewe, especially in the dark or when mingling with other ewes

and lambs. At the same time, in response to maternal care, lambs perform a series of

behaviors after birth initiated by shaking the head, rolling onto the sternum, bleating, pushing

up on the knees and then attempting to stand (Dwyer, 2003; Dwyer, 2008a; Fraser & Broom,

1997). Most newborn lambs are able to stand within the first half hour following birth and

sucking occurs within the first 2 hours of birth in normal cases (Dwyer, 2003). Studies have

shown that the rate of lamb survival is higher in lambs that stand and suckle quickly after

birth (Dwyer et al., 2005). However, the lamb’s first attempts to suck are usually

unsuccessful. There are a number of known factors affecting the behavior in the lamb

including breed, sex, litter size, prenatal nutrition, placental insufficiency, the ewe’s parity

and the birth process (Dwyer, 2003; Dwyer et al., 2005). Male lambs tend to be slower than

6

females to rise on their knees and to attempt to suck, whereas triplets are slower than twin or

single lambs to suck (Dwyer, 2003; Dwyer et al., 2005). Likewise, using an actigraphy

method, a previous study in our lab also found that from postnatal day 2 to 4 male lambs

seem to be less active than females but the situation was reversed from day 5 to 9 (Rurak et

al., 2008). Bonding is also developed between twins or triplet lambs. They seem to stay

closer to each other than to any alien lambs. Such attachment is established through visual

cues and voice recognition (Fraser & Broom, 1997).

1.1.4 Hypothalamic-Pituitary-Adrenal (HPA) Axis Function:

The fetal HPA axis plays an essential role in fetal development, maturation,

homeostasis and even neonatal survival. Studies have shown that the timing of HPA axis

maturation is species-specific and depends on the timing of the brain growth spurt. In

precocious animals such as sheep, the brain growth spurt, which is associated with a large

proportion of neuroendocrine maturation (including corticosteroid receptor development),

takes place in utero (Dobbing & Sands, 1979; Matthews et al., 2002).

It had been well established that the plasma concentration of cortisol, a final product

of the HPA axis, increases markedly during the last 5-10 days of pregnancy and can actively

initiate the onset of parturition by changes in the progesterone/estrogen ratio (Challis et al.,

2000; Mellor, Matheson, & Small, 1977; Norman et al.,1985; Thorburn & Challis, 1979).

Therefore, fetal hypophysectomy or adrenalectomy prolong pregnancy (Liggins, Holm, &

Kennedy, 1966), whereas infusions of adrenocorticotrophin (ACTH) or glucocorticoids can

cause premature delivery (Liggins, 1968; Thorburn & Challis, 1979). The prepartum surge of

7

cortisol is considered as a preparation for the fetus to face with stressful events at birth and

enable them to adapt to extrauterine life (Mastorakos & Ilias, 2003).

Postnatally, plasma cortisol concentration decreases markedly during the first 1-2

hours after birth and continues to decline for 24h thereafter (Mellor & Pearson, 1977). The

circadian rhythm of plasma cortisol does not appear until the third week of age in sheep. This

appearance of a 24 h rhythm of cortisol is not influenced by the presence of a light: dark

cycle since it appears at the same time in newborn lambs raised both under 12:12 hour light:

dark or constant light condition (Parraguez et al., 1989).

Sex differences in the HPA axis function has been observed and attributed to the

differences in the levels of gonadal hormones such as estrogens, progesterone, testosterone

(Carey et al., 1995; Viau & Meaney, 1991; Young, 1995; Young, 1996). This could

contribute, at least in part, to the higher prevalence of depression in females than in adult

males (Brummelte & Galea, 2009). In rats, estrogen enhances HPA axis responses (Viau &

Meaney, 1991), whereas androgen depresses HPA axis activity (Viau, 2002). In sheep, sex

differences in cortisol response only depend on the type of acute stressors but not the gonadal

status (Turner et al., 2002).

1.1.5 Renal Function:

Previous studies have shown that renal function changes from the fetal to the

newborn period in lambs, which indicates the rapid adjustment of the kidney to the changes

in fluid and electrolyte supply. The glomerular filtration rate (GFR) increases from 4.59 ±

0.27ml/min to 6.94 ± 1.0 ml/min, whereas urine flow, sodium and osmolar excretion

decreased and urinary osmolarity increased (more concentrated urine) in newborn lambs 24 h

8

following birth compared to fetal lambs (Smith & Lumbers, 1989). Renal adaptations in the

newborn lambs appear to be independent of their gestational age (Berry et al., 1995). The

mechanisms responsible for the increase in GFR in the postnatal lambs include an elevated

net filtration pressure and ultrafiltration coefficient (Turner et al, 2008). In fetal sheep, acute

stress due to surgery can reduce urine flow (Gresham et al, 1972), which can also be the case

with birth events. Given that urine flow is influenced by water and fluid intake, GFR and

sodium reabsorption, dietary changes from milk-fed to solid intake at the time of weaning are

hypothesized to cause a decline in urine flow rate.

1.2 Depression: Symptoms And Diagnosis:

Depression is often referred to as being one specific form of a mental health problem;

however there are actually several categories of depressive illnesses. The most common form

of depression is Major Depression or Major Depressive Disorder. This form of depression

comprises a combination of symptoms that interferes with one’s ability to work, study, sleep,

eat and enjoy activities that are pleasurable. A less severe form of depression is dysthymia, in

which the long-lasting symptoms do not seriously disable one, but keep one from functioning

well or feeling good. And another form of depressive illness is Bipolar Disorder, which is

characterized by cycling mood changes: high (manic episodes) and low (depressive episodes)

(Dubovsky et al., 2003).

The persistence of symptoms is a key to diagnosis. Many of us might feel blue or

down several times in our life, especially, for example when we lose a loved one. Sometimes

we may even have passing thoughts of suicide. However, in order to differentiate between

real depression and these more commonplace situations, we need to consider the time frame

9

of those feelings or symptoms. Based on the DSM-IV, diagnosis for major depression

disorder is made if symptoms are present for at least 2-week duration (table 1, criteria for

diagnosis of depression, DSM-IV). Conversely, dysthymia is considered as chronic

depression in which the symptoms must persist for at least 2 years to meet the diagnostic

criteria.

Assessing depression in children and youth is different from adults because the

symptoms may be atypical in these populations; for example, depressed mood in children and

adolescents can present as an irritable mood, or the weight loss criteria queried in adults can

be reconsidered as a failure to make the expected weight gain in children. In dysthymic

disorder, the time period for diagnosis is also shorter for children, at least one year (instead of

2 years in adults).

10

Table 1.1: DSM-IV-TR criteria for major depressive episode

A. Patients must have experienced at least five of the nine symptoms below, for the

same 2 weeks or more, for most of the time almost every day. One of the symptoms must

be either (a) depressed mood, or (b) loss of interest.

a. Depressed mood

b. A significantly reduced level of interest or pleasure in most or all activities.

c. A considerable loss or gain of weight (5% or more change of weight in a

month when not dieting). This may also be an increase or decrease in appetite

d. Difficulty falling or staying asleep (insomnia), or sleeping more than usual

(hypersomnia)

e. Behaviour that is agitated or slowed down.

f. Feeling fatigued, or diminished energy

g. Thoughts of worthlessness or extreme guilt (not about being ill)

h. Ability to think, concentrate, or make decisions is reduced

i. Frequent thoughts of death or suicide (with or without a specific plan), or

attempt of suicide

Adapted from Quick reference to the Diagnostic criteria from DSM-IV-TR, American

Psychiatric Association (Mood disorders. 2000).

1.3 Pathophysiology Of Depression:

Many theories have been proposed for the pathophysiology of affective illness. In

1965, extrapolating the indirect evidence from the pharmacological studies, Schildkraut

11

proposed the catecholamine hypothesis of affective disorders. This hypothesis states that

depression is associated with a decrease in catecholamines, particularly norepinephrine,

available at central adrenergic receptor sites (Schildkraut, 1965). On the other hand, the

indoleamine theory postulates that it is not only noradrenaline but also a serotonin deficiency

that contributes to the disrupted regulation of basic behaviors observed in depressed patients.

In addition, studies have shown that an intact serotonin system is necessary for optimal

functioning of noradrenergic neurons (Price et al., 1990). Subsequently, the roles of

cholinergic-noradrenaline systems in depression were emphasized in the work of David

Janowsky et al (Janowsky et al., 1972). Dopamine also appears to play a role in the

pathophysiology of depression. Decreased dopamine transmission has been associated with

depression in post-mortem and animal studies (Dunlop & Nemeroff, 2007; Nutt et al., 2006).

Collectively, these theories are termed the monoamine hypothesis, as they involve

deficiencies in norepinephrine, dopamine and serotonin.

Altered brain serotoninergic function has been implicated as an underlying cause of

not only depression but also of other psychiatric disorders including anxiety, obsessive-

compulsive disorder, eating disorders and substance dependence (Bellivier et al., 1998;

Lucki, 1998; Mann et al., 2001). Evidence that supports the serotonin theory includes: (a)

significantly lower plasma tryptophan levels in subjects with major depression as compared

to healthy controls (Coppen et al., 1973; Cowen et al., 1989), (b) lower cerebrospinal fluid

concentrations of 5-hydroxyindoleacetic acid (5-HIAA), a major metabolite of 5-hydroxy-

tryptophan (5-HT), in untreated depressed patients (Owens & Nemeroff, 1994), (c) lower

concentrations of 5-HT and 5-HIAA in postmortem brain tissue of depressed and/or suicidal

subjects (Owens & Nemeroff, 1994), (d) a decrease in the number of 5-HT transporter or

12

binding sites in postmortem brain tissues of depressed patients and suicide victims and in

platelets of untreated depressed patients (Owens & Nemeroff, 1994). In addition, depriving

the brain of tryptophan, which leads to a central serotonin deficiency, can induce depression

within hours (Lam et al., 1996) or a profound relapse in remitted depressed patients who

previously responded to serotonergic antidepressant therapy (Owens & Nemeroff, 1994).

It is well-known that the essential amino acid tryptophan is a precursor for serotonin

(5-HT) synthesis in the human body. Once ingested, tryptophan is converted to 5-HT through

a series of reactions. However, the majority of tryptophan is converted to kynurenine

following the pathway as described in Fig. 1.1. A portion of serotonin is also converted to

melatonin. (Fig. 1.1) (Jonnakuty & Gragnoli, 2008; Kopin et al, 1961; Myint et al., 2007;

Watts et al, 2012)

5-HT is found in both the central nervous system, mostly in the brain-stem neurons of

the raphe nuclei, and in the peripheral system, mostly in the enterochromaffin cells in the

gastrointestinal tract crypts (Furness & Costa, 1982; Maurer-Spurej et al., 2004). After

release into the blood circulation from the enterochromaffin cells, 5-HT is rapidly taken up

by platelets via the 5-HT transporter and is stored in platelet dense granules. Therefore,

peripherally, platelets store almost all the total body circulating serotonin (Maurer-Spurej et

al., 2004). Since the blood brain barrier is impermeable to peripheral 5-HT, central 5-HT

synthesis depends on the amount of tryptophan or 5-hydroxy-L tryptophan available

peripherally to cross the blood brain barrier. While tryptophan has to compete with other

amino acids such as valine, leucine and isoleucine for a carrier protein in order to cross the

blood brain barrier, 5-hydroxy-L tryptophan can easily cross the blood brain barrier since it

does not require a specific transport protein (Rahman et al., 1982; Yuwiler et al., 1977).

13

Centrally, 5-HT functions at the neuron synapses and influences a variety of behavioral,

physiological and cognitive functions such as memory, mood, emotions, wakefulness, sleep,

appetite and temperature regulation (Jacobs & Azmitia, 1992).

In the periphery, traditionally as its name suggests, serotonin is a vasoconstrictor

causing hypertension (Maurer-Spurej et al., 2004). On the other hand, it also mediates nitric

oxide release and functions as an endothelium-derived relaxing factor (Maurer-Spurej et al.,

2004). It exerts its action through many subtypes of 5-HT receptors present on vascular

smooth muscle cells and endothelial cells (5-HT1B, 5-HT 2A, 5-HT 2B, 5-HT 4 and 5-HT 7)

(Nilsson et al., 1999). 5-HT is metabolized primarily in the liver and only a small fraction

escapes from this primary metabolism and is metabolized in the endothelium of lung

capillaries. Finally, the product of 5-HT metabolism, 5-Hydroxyindoleacetic acid (5-HIAA),

from either central or peripheral sources, is excreted in the urine (Green & Curzon, 1968).

In addition to the indolamine theory of depression, collective evidence has suggested

a corticosteroid receptor hypothesis in depression (Holsboer et al., 1984; Holsboer, 1999;

Holsboer, 2000). Overproduction of corticotrophin releasing hormone which causes an

excess activity of the HPA axis with hypercortisolemia is also observed in many depressed

patients (Gillespie & Nemeroff, 2005; Vreeburg et al., 2009). This prolonged or excessive

secretion of glucocorticoids may lead to suppression of neurogenesis and hippocampal

atrophy (Belmaker & Agam, 2008; Heim & Nemeroff, 2001; Sapolsky, 2000).

In parallel, the leptin hypothesis of depression has also been proposed based on

supporting evidence from animal and human studies. (Lu et al., 2007) The hypothesis of

leptin insufficiency in depression is mainly supported by data from a rodent study. Decreased

14

plasma leptin is associated with behavioral changes in chronically stressed mice or rats and

systemic administration of leptin can reverse the chronic stress-induced behavior changes or

depressive-like behaviors (i.e a decrease in sucrose preference or reduced duration of

immobility in force swim test or tail suspension test) together with an increase in c-fos

expression in the hippocampus. (Lu et al., 2006; Lu et al., 2007) Thus, leptin acts as an

antidepressant in the rat model. However, in the human, leptin insufficiency seems to occur

in only a subpopulation of depressed patients and it alone cannot explain the association

between obesity and depression. Thus, it raised a complementary hypothesis of leptin

resistance, which is similar to insulin resistance in type 2 diabetic patients. In addition, since

leptin is also capable of modulating HPA axis function, the leptin hypothesis for depression

might be complementary to the aboved mention HPA hypothesis of depression. (Lu et al.,

2007)

15

FIG. 1.1: Biosynthesis and metabolism of serotonin.

(Jonnakuty & Gragnoli, 2008; Kopin et al, 1961; Myint et al., 2007; Watts et al, 2012)

Kynurenine

N-acetylserotonin

Melatonin

Indoleamine diooxygenase/Tryptophan dioxygenase Tryptophan (TP)

5-hydroxy-tryptophan (5-HTP)

5-hydroxy-tryptamine (5-HT)

5-hydroxyindole acetaldehyde

5-hydroxyindole acetic acid (5-HIAA)

Tryptophan hydroxylase

L-amino acid decarboxylase

Monoamine oxidase

Aldehyde dehydrogenase

Hydroxyindole-O-methyltransferase

16

1.4 Depression In Pregnancy, Postpartum And In Children:

1.4.1 Incidence:

Even though depression can occur in both genders, it affects more women than men

with a female to male ratio of 1.7-2.7 (Bhatia & Bhatia, 1999; Burt & Stein, 2002; Kessler et

al, 1993). It is believed that biologic factors such as endocrine, genetic or social features

contribute to this gender difference (Nonacs & Cohen, 2003; Weissman & Olfson, 1995).

Studies have shown that acute depletion of tryptophan has a more profound impact on mood

in women as compared to men. This implies that women and men are different in terms of

the magnitude and functional activity of their central serotonergic system (Nishizawa et al.,

1997). Additionally, tryptophan pyrrolase, an enzyme that reduces blood tryptophan levels,

which is an amino acid precursor of serotonin, may be stimulated by circulating

concentrations of estrogen and progesterone (Khan et al.,, 2005). This may, in part, account

for the greater frequency of depression among women during their childbearing years.

Whatever the cause might be, women are much more likely to suffer from depression at any

stage of their lifetime, especially during their childbearing and child-rearing years. The

incidence of clinical depression during pregnancy is about 10-15% (Bhatia & Bhatia, 1999;

Burt & Stein, 2002; Nonacs & Cohen, 2003). Postpartum depression occurs in 10-22% of

women (Burt & Stein, 2002), with symptoms beginning two weeks after delivery including

tearfulness, despondency, labile mood, feelings of inadequacy and inability to cope,

particularly with the baby (Cooper & Murray, 1998; Hopkins et al., 1984; Murray, 1992;

O'Hara, 1997).

17

Depression in children and adolescents is an important issue as depressed adolescents

frequently grow up to be depressed adults (Emslie et al., 1997; Harrington et al., 1990;

Lewinsohn et al., 1999; Rao et al., 1999; Weissman et al., 1999). They have more social

problems later in life at work and with their families, attempt suicide more often and require

more medical and psychiatric attention (Birmaher et al., 1996; Brent et al., 1988; Rohde et

al., 1994). Suicide is the fourth leading cause of death in 10 to 14 year old children and rises

to the third leading cause of death in adolescents (Anderson, 2002). Statistics in Canada

reported that the suicide deaths in 1996 have increased three fold in 10-14 age group as

compared to the rate in 1971 (Houle & Wilkins, 2010). In addition, there is a subsequent

increase in the likelihood of tobacco use, involvement in deviant activities and accidents and

impaired relationship with parents and partners, and also impaired academic function in these

depressed adolescents (Birmaher et al., 1996; Kandel & Davies, 1986; Rohde et al., 1994).

Childhood depression is more common than it used to be thought. In fact, depression

can be diagnosed in children as young as 3 years old (Dubovsky et al., 2003). In general,

depression occurs in 2% of prepubertal children and in 3-8% of adolescents (Birmaher et al,

1998; Costello et al., 1996; Lewinsohn et al., 1994).

1.4.2 Risk Factors Of Depression:

Generally, multiple factors may contribute to the occurrence of depression including

marital status, socioeconomic factors, residence, seasonal and geographic factors, social

stressors, lack of social support and the presence of comorbidity (Kaplan & Sadock's

comprehensive textbook of psychiatry2005). Omega-3-fatty acid, which reduces the turnover

of membrane phospholipids, is considered as an adjunctive mood stabilizer (Dubovsky et al.,

18

2003). However, there is evidence that a diet low in omega 3 fatty acids or fish consumption

is not associated with depressed mood, major depression or suicide (Hakkarainen et al.,

2004).

Early experience of loss or trauma and stress can initiate or exacerbate neurochemical

imbalance in vulnerable subjects. This idea is supported by studies in animals in which early

separation from peers and inescapable situations profoundly alters the turnover of their

biogenic amines and postsynaptic receptor sensitivity (Gilmer & McKinney, 2003). In

humans, a polymorphism of the serotonin transporter gene can identify who among

traumatized children will develop adult depression (Caspi et al., 2003; Kendler &

Karkowski-Shuman, 1997; Lesch et al., 1996; Ogilvie et al., 1996; Ogilvie & Harmar, 1997).

There are many factors that can play a role in the occurrence of postpartum

depression. Even though there are no firm causal links, the significant changes in hormone

levels of progesterone, estrogen, cortisol, neurosteroids and β-endorphins may lead to

depression in this period (McCoy et al., 2003; Weissman & Olfson, 1995). Additional

predictive factors are obstetric pregnancy complications, infant irritability and poor motor

function in the early neonatal period (Murray, 1993; Murray et al., 1996; Waterstone et al.,

2003). Besides these, probably the most attributed risks are psychiatric and psychosocial

factors including prior history of mood disorder, low socioeconomic status, lack of partner or

family support, an unwanted pregnancy and young maternal age (Burt & Stein, 2002;

Hopkins et al., 1984; Murray, 1993; Warner et al., 1996).

When left untreated, depression during pregnancy can lead to changes in maternal

behaviours such as decreased appetite, poor pregnancy-related weight gain, increased

19

likelihood of smoking and/or alcohol and drug consumption and even a risk of suicide in

severe cases (Ahluwalia et al., 2004; Bhatia & Bhatia, 1999; Nonacs & Cohen, 2003). There

is also evidence that these poor maternal health behaviours or depression per se are

associated with an increased risk of ischemic heart disease (Glassman & Shapiro, 1998).

These place both the mother and fetus at risk.

1.4.3 Effects Of Maternal Depression On Fetal Neurobehavioral Development:

Studies have shown that depression per se, without poor maternal behavior, can result

in pregnancy complications such as preterm birth and/or small for gestational age, and

increased rates of maternal obstetric complications (Anderson, 2004; Andersson et al., 2004a;

Andersson et al., 2004b; Dole et al., 2003; Hoffman & Hatch, 1996; McCormick et al., 1990;

Orr & Miller, 1995; Orr et al., 2002; Peacock et al., 1995; Steer et al.,, 1992; Weissman &

Olfson, 1995). It is not yet clear via which mechanisms depression can lead to preterm birth

and fetal growth restriction. One explanation for this relationship is that the elevated plasma

concentrations of maternal cortisol and catecholamines associated with depression lead to a

decrease in uterine blood flow (Hoffman & Hatch, 1996; Lake et al., 1982; Linkowski, 2003;

Weissman & Olfson, 1995; Rurak, 1995), and thus reduce nutrient supply to the fetus and

impair fetal growth (Lang et al., 2000). Moreover, the elevated endogenous maternal

corticosteroids and catecholamine concentrations can affect the fetal central nervous system

causing infant irritability such as excessive crying, difficulty in consoling, which is a

predictive factor of postpartum depression (Murray, 1993; Welberg et al., 2000; Zuckerman

et al.,1990). As a result, maternal depression during pregnancy can extend into the

postpartum period via altered neonatal behaviour (Zuckerman et al., 1990). This, in turn,

could further impair neurological development of the offspring. Indeed, some evidence has

20

indicated that maternal depression can adversely affect temperament and cognitive

development in the infant. Similarly, depressed mothers of preschoolers have more negative

perceptions of and interactions with their children (Beck, 1996; Hay & Kumar, 1995; Lang et

al., 1996).

1.4.4 Treatment Of Depression:

Given the negative impact of maternal depression in pregnancy on both the mother

and offspring, treatment for this condition is essential. Due to the general concern about

exposure of the fetus to drugs, psychotherapy seems to be a preferable choice of treatment.

However, there are few studies that assess the efficacy of psychotherapy during pregnancy.

Drug therapy is a norm and antidepressants are the preferred course of treatment even though

these drugs are not approved for use in pregnancy (Altshuler et al., 2001; Bhatia & Bhatia,

1999; Nonacs & Cohen, 2003; Weissman & Olfson, 1995). Selective serotonin reuptake

inhibitors (SSRIs) were first introduced into the market in the late of 1980s (DeVane, 1999),

and by 1998 they largely replaced the older classes of antidepressants for the treatment of

depression in pregnancy (Altshuler et al., 2001; Goldstein, 1998; Goodnick & Goldstein,

1998b; Misri et al., 2000; Nonacs & Cohen, 2003; Simon et al., 2002; Skop et al., 1994) due

to their lower incidence of side effects and greater safety in terms of drug overdose

(Goodnick & Goldstein, 1998b; Jones & Blackburn, 2002). One recent study has reported an

increasing use of antidepressants in pregnancy from 2% in 1996 to 7.6% of deliveries in

2004-2005, and in which SSRI use increased 4.9% from 1996 to 2004 (Andrade et al., 2008).

There are similar reports that SSRI accounts for more than 70% of prescribed antidepressant

as a pharmacotherapy in pregnant women (Cooper et al., 2007; Simon et al., 2002). In

addition, these agents have also been used to treat other psychotic disorders in pregnancy,

21

which are associated with a dysfunctional serotonin system, such as anxiety and obsessive-

compulsive disorders (OCD) (Levine et al., 2003; Vaswani et al., 2003).

1.5 Selective Serotonin Reuptake Inhibitors (SSRIs):

1.5.1 Clinical Uses:

SSRIs are considered as a second generation of antidepressant after the first

generation of monoamine oxidase inhibitors (MAOI) and tricyclic antidepressants (TCA). In

general, SSRIs are widely used in psychiatry. Since SSRIs are relatively safe in terms of

overdose and have a more benign side-effect profile than the older generation agents, they

offer a greater tolerability and compliance to the treatment. (Lane et al., 1995; Masand &

Gupta., 1999) Besides their main indication for the treatment of major depressive disorders,

SSRIs are also prescribed as a first-line pharmacological treatment for other disorders such as

bipolar disorder, social anxiety disorder, generalized anxiety disorder, obsessive-compulsive

disorder (in combination with cognitive-behavioral therapy), panic disorder, eating disorder

(bulimia), premenstrual dysphoric disorder, and posttraumatic stress disorder. (Boyer et al.,

1992, Masand & Gupta., 1999) In addition, they are also used to control mood disorders in

special subsets of the population including post stroke patients, and terminally-ill patients

(cancer, HIV/ADIS) (Franco-Bronson et al., 1996; Sauer et al., 2001) A typical response to

treatment occurs within four to six weeks of SSRIs treatment.

1.5.2 Mechanism Of Action:

Monoamine transmitter activity may be regulated at many different levels including

synthesis, packaging and storage of neurotransmitters in synaptic vesicles, release, reuptake,

22

metabolism and postsynaptic receptor responsiveness (Fig. 1.3) (Kaplan & Sadock's

comprehensive textbook of psychiatry 2005).

FIG. 1.2: Schematic diagram of monoaminergic synapse and steps involved in synaptic transmission: Monoamine neurotransmitters (1) are synthesized within neurons from common amino acid precursors, which are Tryptophan and 5-Hydroxytryptophan (5-HT) in the case of serotonin, (2) are taken up into synaptic vesicles via a vesicular monoamine transporter, (3) are released into the synaptic cleft upon stimulation, (4) interact with postsynaptic receptors to alter the excitability of postsynaptic cells, (5) interact with presynaptic autoreceptors located on the nerve terminal to suppress further release. (6) Released monoamines may also be taken back up from the synaptic cleft into the nerve terminal by plasma membrane transporter proteins. (7) Once taken up, monoamine may be subject to enzymatic degradation or may be protected from degradation by uptake into vesicles. MAO: monoamine oxidase Adapted from Kaplan and Sadock’s comprehensive textbook of Psychiatry 2005.

1

2

3

4

5 6

7

MAO

amino acidprecursor

monoamine neurotransmitter

23

The goal of drug therapy in the treatment of depression is to maintain the circulating

neurotransmitter concentration in the synapse at a certain level, either by increasing the

amount of neurotransmitter produced for release or the duration of time the neurotransmitter

spends in the synapse. Antidepressants are classified based on the type of monoamine

neurotransmitter (dopamine, noradrenaline or serotonin), being affected at the synapse and

the specific mechanisms being impacted such as synthesis, release, reuptake, and

metabolism. There are three major classes of antidepressants. Monoamine oxidase inhibitors

(MAOI) prevent the degradation of noradrenaline and serotonin in the presynaptic cell.

Tricyclic acids (TCA) block the reuptake of noradrenaline and serotonin to varying degrees

at the presynaptic cell. SSRIs selectively inhibit the reuptake of serotonin (5-HT) by

presynaptic serotoninergic neurons and peripheral serotonin containing cells, particularly

platelets, via effects on the serotonin transporter (Anderson, 2004; Goodnick & Goldstein,

1998a; Stahl, 1998; Wright & Angus, 1989). In addition, there are sub-classes of dual

inhibitors of the three neurotransmitters mentioned above that have been developed,

including norepinephrine/dopamine reuptake inhibitor (NDRI), serotonin/norepinephrine

reuptake inhibitor (SNRI), noradrenergic and specific serotonergic antidepressant (NaSSA)

and serotonin antagonist reuptake inhibitor (SARI) (Stahl, 1998).

The various SSRIs (sertraline, fluoxetine, paroxetine, citaloprame, fluvoxamine)

differ in their potency and inhibitory effects on reuptake of other monoamine

neurotransmitters (Kaplan & Sadock's comprehensive textbook of psychiatry 2005).

However, comparisons of these agents in acute treatment have shown that they have similar

efficacies, probably due to the parallel differences in dose and plasma concentration

(DeVane, 2003; Hiemke & Hartter, 2000).

24

After SSRI treatment, the inhibition of serotonin reuptake usually occurs immediately

whereas the onset of the antidepressant effect is delayed for about 2-6 weeks (Goodnick &

Goldstein, 1998b). It is generally accepted that the initial rise in extracellular 5-HT in the

somatodendritic regions of the dorsal and median raphe nuclei activates somatodendritic 5-

HT1A autoreceptors which are responsible for the inhibition of the firing of 5-HT neurons

and thus reducing 5-HT release. Yet, when given chronically, SSRI will down-regulate the 5-

HT1A autoreceptors and increase serotonergic neurotransmission (Celada et al, 2004;

Hensler, 2003; Hjorth, 1996; Stahl, 1998). In addition, other neurotransmitters can also be

involved in the control of the firing rate and serotonin release (Kalsner, 2000; Kalsner &

Abdali, 2002).

1.5.3 Pharmacokinetics:

The different SSRIs vary in their half-lives and other pharmacokinetic variables.

Fluoxetine (FX) has the longest half-life, whereas the values for the other SSRIs are shorter

(Hiemke & Hartter, 2000; Vaswani et al., 2003). The wide difference in the half-lives among

SSRIs account for the differences not only in the time to reach the steady state, the time to

clear the drug from the body after discontinuation but also in the potential for withdrawal

symptoms after discontinuing the SSRIs. Concomitant use of SSRIs and other agents

metabolized by the cytochrome P450 (CYP) isozymes can lead to drug-drug interactions,

because SSRIs inhibit the activity of some of the isozymes. Among SSRIs, FX and

paroxetine are strong CYP2D6 inhibitors, and the others are weak CYP2D6 inhibitors. FX is

also a moderate CYP2C19 inhibitor (Harvey & Preskorn, 2001). Additionally, FX is also a

substrate of CYP2C19 and CYP2D6 (Jeppesen et al., 1996; Ring et al., 2001; Spina et al.,

2003, Harvey & Preskorn, 2001)

25

Genetic polymorphisms have been identified in two CYP isoenzymes, CYP2D6 and

CYP2C19, and that divides the population into several groups including ultra extensive,

extensive, intermediate and poor metabolizers. The poor metabolizers with nonfunctional

allele of CYP2D6 gene have a severely impaired catalytic function of the isoenzymes. The

clinical impact of poor metabolizers is that they are unable to metabolize the drugs, which are

substrates of these enzymes; therefore the sub-population will have a higher risk of adverse

effects, especially when no other metabolic route is available for the drugs or when multiple

drugs share the same CYP in their metabolism (Vaswani et al., 2003). This becomes more

important when switching medications having a long half live (eg: FX) to other medications

metabolized by the same cytochrome since the risk of toxicity still persists long after the

drugs were discontinued.

Among the SSRIs, paroxetine, sertraline and fluoxetine are highly bound to plasma

proteins and only a small fraction of the drugs stays in the form of free drug. Thus,

coadministration of the SSRIs and another highly-protein-bound drug might elevate the free

concentrations of the other drug, potentially resulting in adverse events.

All of the SSRIs can readily cross the placenta in humans, rats and sheep because

they are highly lipophilic (Hendrick et al., 2003; Kim et al.,2004; Kim et al., 2006; Pohland

et al., 1989; Rampono et al., 2009). SSRIs are also excreted in breast milk. Despite this fact,

the concentrations of the drugs in an infant are much less than those in a fetus, because of the

much lower amount of drug acquired by the infant via breast milk compared to that acquired

by the fetus via placental transfer and also because of the progressive development of

metabolic capacity in the infant following birth (Kim et al., 2006). Hence, the exposure of a

fetus to the drugs during prenatal period is more likely to be the cause of any long-term

26

effects of the drugs in compared to the exposure of an infant to the drugs during the postnatal

period.

1.5.4 Side Effects:

As a class, SSRIs, clearly demonstrate superiority in terms of safety and tolerability

compared to the older antidepressant medications. A meta-analysis of 84 double-blind

Randomized Controlled Trials (RCT) of the adverse effects of antidepressants, particularly

SSRIs and TCA has found that all antidepressants cause adverse effects (Trindade et al.,

1998). Typical anticholinergic side effects, including dry mouth, constipation, blurred vision,

urinary retention and postural hypotension, which are usually reported in tricylic

antidepressants, occur less frequently with SSRIs. Serotonin has been implicated as playing

roles in gastrointestinal motility, appetite control, sexual behavior, sleep regulation, and

cerebral vasomotor tone; therefore SSRIs are predicted to cause nausea, appetite loss, sexual

dysfunction, and sleep disturbances (Dubovsky et al., 2003). In fact, there is evidence that

fluoxetine, fluvoxamine, paroxetine and sertraline have been associated with nausea,

diarrhea, insomnia, nervousness, agitation and anxiety. Even though paroxetine was found to

induce less nausea than the other SSRIs, the difference was not significant (Trindade et al.,

1998). SSRI-associated adverse effects seem to be related to drug dose (Beasley et al.,1991).

Together with the introduction of SSRIs and the increased incidence of depression in

children and adolescents, SSRIs is also used to treat children with this condition. SSRIs were

found to be associated with a higher rate of agitation and suicidal thoughts, or both, from

clinical trials comparing SSRIs treatment and placebo in children and adolescents with major

depressive disorder. Thus, a warning for a close observation of signs or symptoms of

27

worsening depression, including suicidal ideation, early on in therapy with these medications

or on titration of dosing, was required to be included in their labels and product inserts

(Bridge et al., 2007; Hammad et al., 2006). In the meta-analyses, SSRI treatment had a

benefit compared to placebo treatment, but also was associated with significant risks for

adverse events, including suicidal behaviour (except for FX) (Jureidini et al., 2004;

Whittington et al., 2004)

1.6 Fluoxetine (FX):

FX is an antidepressant that belongs to the SSRI class. It was one of the first SSRIs to

be developed. FX (trade name Prozac) was first introduced into the US market in 1988

(Borys et al., 1990). It has been shown to effectively relieve the symptoms of major

depression and is better tolerated than the older classes of antidepressant such as TCA (Cohn

& Wilcox, 1985). Currently, FX also has received approved indications for OCD, bulimia

nervosa and premenstrual dysphoric disorder. In 2003, the FDA approved the combination of

FX and olanzapine (trade name Symbyax) for treatment-resistant depression (unresponsive to

two separate trials of different antidepressants). Despite the fact that many new SSRI agents

have been introduced into the market after FX was, FX remains very popular. In 2008, over

23 million prescriptions of FX were filled in the US, an increase of 4.5% from 2007. Also, it

was ranked the second most prescribed antidepressant (Top 200 generic drugs by total

prescriptions 20082009).

28

1.6.1 Pharmacokinetics:

1.6.1.1 Absorption:

The bioavailability of FX is relatively high, about 70 to 90 percent. This

bioavailability results from an almost complete absorption and a limited hepatic first-pass

effect. The time to maximum concentration (tmax) of FX is about 6-8 hours. And the peak

plasma concentration (Cmax) of FX is about 15-55 ng/ml (Altamura et al., 1994). Food does

not decrease the absorption, but may delay the time to reach the peak concentration by 1-2

hours (Benfield et al., 1986).

1.6.1.2 Distribution:

FX is one of the SSRIs highly bound to plasma proteins, approximately 95%. The

volume of distribution (Vd) of FX and its active metabolite, norfluoxetine (NFX), falls in the

range from 12 to 45 L/kg (Benfield et al., 1986).

1.6.1.3 Metabolism:

FX is a 1:1 racemic mixture of two optical isomers. Both S- and R- enantiomers have

equivalent pharmacologic activity, although the S-enantiomer is eliminated more slowly and

therefore presents predominantly in the plasma at steady state (Wong, Fuller, & Robertson,

1990). FX is metabolized by the cytochrome (CYP) P450 isozymes in the liver. FX is

demethylated to its major active metabolite, norfluoxetine (NFX) and several inactive

metabolites. The two clinically important CYP P450 isozymes involving the metabolism of

FX are CYP2C19 and CYP2D6 (Stevens & Wrighton, 1993). CYP2D6 catalyzed the

metabolism of R & S FX and S-NFX but not of R-NFX (Fjordside et al., 1999).

29

1.6.1.4 Elimination:

Hepatic metabolism of FX to its metabolites then renal excretion is the primary route

of elimination. The half-life (t1/2) values of fluoxetine following an acute and chronic

administration are 1-3 days and 4-6 days respectively. The major active metabolite, NFX, has

a longer half-life, which ranges from 4 to 16 days, independent of length of treatment. The

relatively slow elimination of FX and NFX ensure that they will remain in the body for

weeks after discontinuation. Hepatic impairment will prolong the half-life elimination of FX

and NFX.

1.7 FX Effects On Newborns And Infants:

1.7.1 FX Effects On Cardiovascular Functions:

There are a few case reports in adults of adverse cardiac events after starting FX

therapy including sinus bradycardia, supraventricular tachycardia, atrial fibrillation, atrial

flutter and ST segment depression. (Ahmed et al., 1993; Gardner et al., 1991; Isbister et al.,

2004; McAnally et al., 1992; Riddle et al., 1989; Roberge & Martin, 1994). One case report

of FX poisoning in which the patient ingested 12g of FX and developed bradycardia,

progressing to ventricular fibrillation and asystole (Compton et al., 2005). Another case

report of QT prolongation to 600msec and torsades de point in a 74 year-old woman occurred

3 weeks after starting FX therapy (Appleby et al., 1995).

QTc interval prolongation was also reported in a newborn after in utero exposure to

FX. To note, a prolonged QTc interval (> 560 msec) predisposes the sufferers to torsade de

pointes, an intermittent ventricular dysrhythmia which, in turn, can lead to a potentially life-

30

threatening monomorphic ventricular tachycardia or fibrillation. A subsequent retrospective

cohort study comparing 52 SSRIs exposed newborns with 52 unexposed controls matched by

gestational age also confirmed that the exposed newborns had a longer QTc interval than the

unexposed controls (409 vs 392 msec, respectively). However, there were no cases of

arrhythmia reported. The SSRIs used in the study included paroxetine, citalopram,

fluoxetine, fluvoxamine and venlafaxine and there was no drug-specific evaluation or serum

electrolytes measurement (Dubnov-Raz et al., 2008). In agreement with these findings,

prolongation of QTc interval of no greater than 450 msec existed in 62% of FX overdose

patients in one study, but none developed ventricular dysrhythmia (Phillips et al., 1997).

Nevertheless, of the SSRIs, FX appears to have minimal cardiotoxicity.

Effects of SSRIs exposure on cardiovascular function are likely to originate in utero.

Recently our group has found a reduced fetal middle cerebral artery (MCA) flow resistance

and cross sectional MCA area and fetal heart rate variability in the SSRIs-exposed fetus,

even after controlling for maternal mood. Importantly, despite a reduced MCA flow

resistance, MCA volume flow was not increased, and this was associated with a reduced

cross sectional MCA area (Rurak et al., 2011).

Chambers et al., (2006) was one of the first papers that reported an increased risk of

persistent pulmonary hypertension (PPHN) development (adjusted OR = 6.1, 95% CI 2.2-

16.8) in newborns whose mothers were taking SSRIs during late gestation. Even though there

were some methodological limitations in the study such as retrospective design and potential

recall bias, her finding was confirmed by a subsequent study by Kallen & Olausson, (2008).

Despite the fact that persistent pulmonary hypertension is a rare condition, these findings

prompted the FDA to issue a public health advisory to add a warning label of potential risk of

31

PPHN in 3rd trimester SSRI-exposed infants (Public health advisory: Treatment challenges

of depression in pregnancy and possibility of persistent pulmonary hypertension in

newborns.2006). However, two following studies, which involved retrospective examinations

of administrative health records, failed to demonstrate this increased risk of PPHN in SSRIs-

exposed infants (Andrade et al., 2009; Wichman et al., 2009). However, both studies were

underpowered to detect small differences in the incidence of PPHN, which itself is a rare

event. In another human study (Wilson et al, 2011), several potential factors, including use of

SSRIs after 20 weeks gestation, were examined in 20 case of primary PPHN compared to

120 controls. The only significant association was between cesarean delivery before labor

onset and PPHN and the authors noted that the 2 studies reporting an association between

antenatal SSRI exposure and PPHN (Chambers et al., 2006; Kallen et al., 2008) did not

control for the mode of delivery. A very recent study from our group measuring fetal right

pulmonary artery resistance, flow and vessel cross sectional area by Doppler ultrasound also

did not support an association between SSRIs exposure and PPHN in the infants even though

the authors observed a significantly higher rate of right pulmonary artery flow in the SSRI-

exposed fetuses with respiratory difficulties (Lim et al., 2012).

In a study in pregnant rats involving daily administration of fluoxetine by gastric

gavage from day 11 to 21 of pregnancy (Fornaro et al., 2007), the offspring from the

fluoxetine treated group exhibited an increase in pulmonary arterial medial thickness and in

the right/left ventricular weight ratio. In vitro, fluoxetine at lower concentrations increased

proliferation of fetal but not adult pulmonary arterial smooth muscle cells, whereas at higher

concentrations the drug inhibited proliferation of both the fetal and adult cells.

32

In sheep, 8 days of late gestational FX infusion in pregnant ewes did not result in any

changes in the daily average maternal arterial pressure and heart rate nor in fetal arterial

pressure. However, there was a transient decrease of fetal heart rate on both the FX exposed

group (infusion d2 and 6) and the control group (infusion day 4 and 8) (Morrison et al.,

2002).

1.7.2 FX Effects On The Homeostatic And Respiratory Functions:

Extensive research focused on brainstem mechanisms underlying sudden infant death

syndrome has shown the essential role of 5-HT neurons in the modulation of the respiratory

response and thus homeostatic functions (Kinney, 2009; Kinney et al., 2009). In vitro studies

have indicated that the midbrain 5-HT neurons, which serve as an arousal chemoreceptor,

and the medullary 5-HT neurons, which function as respiratory chemoreceptor, increase their

firing rate 3-fold on average in response to hypercarnia and a decrease in pH from 7.4 to 7.2

(Buchanan & Richerson, 2010; Richerson, 2004; Wang et al., 1998). In addition, acute

hypercapnia can also activate the limbic systems causing dyspnea and panic which are

usually observed in panic disorders (Gorman et al., 1994; von Leupoldt & Dahme, 2005).

Clearly, given that 5-HT neurons play an important role in homeostatic function, changes in

the serotonergic system either by depression itself or pharmacologic intervention may lead to

changes in homeostatic systems.

1.7.3 FX Effects On Birth Outcomes:

A majority of research regarding the effects of SSRI on birth and pregnancy

outcomes have found that late pregnancy SSRI exposure increases the risk of preterm birth,

shorter gestational length, lower birth weight, smaller than gestational age, lower APGAR

33

score at birth, admission to NICU and reduced postnatal weight gain (Chambers et al., 1996;

Einarson et al., 2010; Kallen, 2004; Lewis et al., 2010; Maschi et al., 2008; Oberlander et al.

2006; Oberlander et al. 2008; Pearson et al., 2007; Simon et al., 2002; Suri et al., 2007; Wen

et al., 2006; Wisner et al., 2009; Zeskind & Stephens, 2004). However, others have failed to

confirm these effects (Casper et al., 2003; Hendrick et al., 2003; Kulin et al., 1998; Lund et

al. 2009; Malm et al., 2005; Suri et al., 2004). There are conflicting findings regarding the

impact of timing and duration of SSRI exposure during pregnancy on perinatal outcomes.

Chambers et al., (1996) found that preterm birth, low birth weight and poor neonatal

adaptation were significantly more common in a late gestational exposure group than in an

early exposure group. Conversely, Oberlander et al., (2008) reported early and late exposure

groups had the same neonatal outcomes while an increase in days of exposure was

significantly associated with low birth weight and low gestational age, even after controlling

for maternal illness and medication dose. Still, others found no association between late

gestation exposure and adverse neonatal outcomes (Cohen et al., 2000; Malm et al., 2005).

The potential reason for the lack of consistency in these findings is the differences in doses

among studies. Recently, it has been reported that much higher preterm birth rate and lower

gestational age were observed in the higher FX dose group, even after controlling for length

of exposure (Roca et al., 2011). The mechanisms underlying the association between prenatal

SSRI exposure and the shortening of gestational length are not yet been clear. However, it

has been proposed that SSRI exposure might promote adverse birth outcome through the

dysregulation of the fetal HPA system or through the vasoconstrictor effects of serotonin

which could decrease maternal placental blood flow, especially in the 3rd trimister (Morrison

et al., 2002; Roca et al., 2011). SSRIs-induced fetal HPA axis disturbances were evidenced

34

by a previous sheep study in our lab showing an increase in the fetal plasma prepartum

cortisol surge and apparent increased sensitivity of the fetal adrenal gland to ACTH after 8 d

maternal FX administration (Morrison et al., 2004). In addition, a relationship between

elevated placental corticotropin-releasing hormone (CRH) levels, which also regulates

cortisol secretion, and a higher risk of preterm birth and fetal growth restriction has been

established in human pregnancies associated with antenatal SSRI therapy (Wadhwa et al.,

2004). Other potential mechanisms concerning the effect of SSRIs exposure on lower birth

weight or smaller than gestational age include a SSRIs-induced alteration in the fetal IGF-

1/GH axis (Davidson et al., 2009) or a reduction in uterine blood flow by 5-HT, a

vasoconstrictor agent, which limits oxygen and nutrient supply to the fetus (Morrison et al.,

2002) or a direct SSRIs-effect on the growing skeleton by exerting their effect on osteoclast

differentiation and formation (Battaglino et al., 2004; Warden et al., 2005).

1.7.4 FX Effects On Neonatal And Child Behaviors:

Accumulating data from case reports and cohort studies have also indicated that late

gestation exposure to SSRIs increases the risk of a variety of neonatal complications termed

“neonatal behavioral syndrome” or “poor neonatal adaptation” (Chambers et al., 1996;

Cohen et al., 2000; Kwon & Lefkowitz, 2008; Mhanna et al., 1997; Oberlander et al., 2004).

According to some studies, the risk can increase up to 3 times (Chambers et al., 1996; Cohen

et al., 2000; Oberlander et al., 2004). Among SSRIs, most reported cases involved FX and

paroxetine exposure. Behavioral and neurologic symptoms vary from mild central nervous

system, motor, respiratory and gastrointestinal signs and metabolic dysfunction (including

irritability, persistent crying, shivering, tremor, restlessness, feeding difficulties, sleep

disturbances) to severe symptoms (including seizures, dehydration, excessive weight loss,

35

hyperpyrexia and the need for intubation). These symptoms usually disappear by the second

week after birth (Moses-Kolko et al., 2005). These findings led Health Canada and the FDA

in 2004 to include a warning in the drug information labeling for SSRI/SNRIs that neonates

exposed to these agents in the late third trimester have developed adverse events (Summary

minutes of the pediatrics subcommittee of the anti-infective drugs advisory committee.2004).

These symptoms are observed in around 10-30% of the exposed newborns (Koren et al.,

2009). Unfortunately, the mechanisms that may account for these complications have yet to

be elucidated. They may be multifactorial, with the severity of the maternal mental illness

playing a major role.

In fact, one of the big challenges to this type of research in humans is to distinguish

between the drug effects and the impact of maternal psychiatric disorders on the occurrence

of neonatal behavioral syndrome. Even though some studies have enrolled mothers with

untreated depression in their studies to adjust for the possible indication confounders, those

are more likely to have less severe psychiatric problems than mothers with treated

depression. Using a propensity score matching approach, Oberlander et al., (2006) have

found that the incidence of symptoms of behavioral syndrome from SSRI exposed infants is

similar to non-exposed infants born to depressed mothers.

Since the symptoms observed in adults with SSRI discontinuation syndrome overlap

with those seen in neonates born to mothers treated with SSRIs, one of the mechanisms

suggested for poor neonatal adaptation syndrome is a withdrawal syndrome. On the other

hand, some symptoms in the neonates are similar to those seen as side effects of SSRIs or

SSRI toxicity associated with elevated 5-HT levels (serotonin syndrome) (Birmes et al.,

2003; Ener et al., 2003). Therefore, it remains unclear whether neonatal symptoms are due to

36

SSRI withdrawal or directly due to SSRI toxicity (Koren et al., 2009; Moses-Kolko et al.,

2005). However, since theoretically the management for these mechanisms is clearly

opposite, there is a compelling need to identify which is the true case. Currently, the

management for SSRI/SNRI poor neonatal adaptation syndrome is mainly symptomatic, with

supportive care only (Koren et al., 2009).

In parallel with the mechanisms suggested from human studies, previous studies on

fetal lambs with chronically 8-day FX-exposure in late gestation in our lab (Morrison et al.,

2001; Morrison et al., 2002; Morrison et al., 2004; Morrison et al., 2005) have suggested

another mechanism: that late gestational SSRIs exposure could alter fetal brain development,

which relates to the importance of serotonin in the maturation of brain pathways. During the

period of FX administration, the incidence of fetal rapid-eye-movement (REM) sleep was

significantly decreased (Morrison et al., 2001). In addition, a study of human fetal behavioral

development also demonstrated an abnormaly high rate of continual bodily activity during

non-REM sleep in SSRI-exposed fetuses (Mulder et al., 2011). This mechanism is further

supported by a subsequent study from our group which showed additive negative effects in

infants of depressed mothers treated with SSRIs as compared to untreated depressed mothers

(Oberlander et al., 2006). Our recent findings of a decreased MCA flow resistance without

increasing MCA volume flow in the fetus exposed to SSRIs (Rurak et al., 2011) adds more

evidence to support this proposed mechanism. Changes in tissue volume in the brain region

perfused by the MCA, which is secondary to changes in brain blood flow, could be due to a

SSRIs-mediated alteration of serotonergic tone. However, whether the disturbed serotonergic

system places any long-term sequelae during postnatal development beyond the exposure

period still needs further investigation. To date, only two studies have reported significant

37

difference in psychomotor development in SSRIs-exposed versus non-exposed infants

(Casper et al., 2003; Mortensen et al., 2003).

1.7.5 FX Effects On The Hypothalamic-Pituitary-Adrenal (HPA) Axis:

HPA axis activity is proposed to be involved in the pathophysiology of depression

since altered levels of corticotropin-releasing factor in the cerebrospinal fluid (Arborelius et

al., 1999) and hypercortisolemia are observed in depressed patients (Holsboer et al., 1995)

and this was normalized with antidepressants (Holsboer & Barden, 1996) possibly by

increasing hippocampal glucocorticoid receptor expression (Yau et al., 2002). The function

of the HPA axis and serotonergic system are highly interrelated. Hypothalamic CRH might

modulate the serotonergic system (Boadle-Biber et al., 1993; Fuller, 1996a). Conversely,

serotonergic neurons in the raphe nucleus can directly stimulate HPA axis function via 5-

HT1A and 5-HT2A receptors in the paraventricular nucleus of the hypothalamus (Fuller,

1996b). Therefore, increases in extracellular 5-HT levels acutely and in serotonergic

neurotransmission chronically by treatment with SSRIs, such as FX, in depressed mothers

could have an impact on fetal HPA activity. As mentioned previously, in our sheep studies,

8-day late gestational FX i.v infusion in pregnant ewes resulted in an augmented prepartum

rise in fetal ACTH and cortisol levels and reduced low voltage rapid eye movement

behavioral state in the fetus (Morrison et al., 2004). Although the mechanisms are not fully

understood, alterations in HPA axis activity in the fetus might be one of the possibilities

underlying negative perinatal outcomes observed in human studies. In addition, this is

particularly important in the context of fetal exposure to excess glucocorticoids either by

prenatal stress or maternal illness (e.g: depression, anxiety disorders) or pharmacologic

agents effects (eg: SSRIs, synthetic glucocorticoids for lung maturation in preterm labor),

38

which can program the HPA axis in ways that it will have negative impacts on mental,

metabolic and cardiovascular health later in life (Lupien et al., 2009; Seckl, 1997; Tamashiro

& Moran, 2010). Interestingly, a recent study from our group, which further examined the

long-term effects of prenatal SSRIs (fluoxetine) on the HPA axis in the postnatal period,

have shown an association between SSRI exposure and reduced basal cortisol levels as well

as an alteration in the HPA stress response pattern (Oberlander et al., 2008). A lower cord

blood cortisol level in SSRIs-exposed neonates was also reported in a subsequent study

(Davidson et al., 2009). Conflicting findings from the sheep and human studies could be, in

part, attributed to the differences in the timing and duration of exposure which, determine the

nature of the offspring adaptive responses and the physiological disturbances in later life

(Chadio et al., 2007).

Sex differences in the HPA axis normalization in response to antidepressant treatment

have been observed (Binder et al., 2009). In rodents, there is a differential HPA axis response

between males and females following acute SSRIs (citalopram). Female mice had a greater

corticosterone response after citalopram administration than males and testosterone treatment

in ovariectomized females eliminated the sex differences (Goel & Bale, 2010). Similar

differences were also reported with sertraline treatment in sheep (Broadbear et al., 2004).

ACTH and cortisol secretion significantly increased in female but not in male after sertraline

s.c injection, independent of circulating steroid hormones. These studies indicate that the

HPA axis of the female is more sensitive to the stimulatory effects of serotonin than that of

the male.

39

1.8 Rationale:

The incidence of clinical depression during pregnancy is about 10-15% (Bhatia &

Bhatia, 1999; Nonacs & Cohen, 2003). Postpartum depression occurs in 10-22% of women,

with symptoms beginning two weeks after delivery (Burt & Stein, 2002). Depression during

pregnancy can place both the mother and fetus at risk (Ahluwalia et al., 2004; Orr & Miller,

1995; Zuckerman et al., 1989), and there is abundant human evidence for adverse effects of

maternal postpartum depression on the cognitive, motor and emotional development of the

offspring (Kurstjens & Wolke, 2001; Murray & Cooper, 1997; Murray et al., 2001). Since

their introduction in the late 1980’s, SSRIs remain a first-line pharmacological treatment for

both depression in pregnancy and in the postpartum period (Altshuler et al., 2001; Misri et

al., 2000). However, numerous studies in humans have shown that infants delivered from

women taking SSRIs during pregnancy have a higher risk of a number of adverse pregnancy

outcomes, including premature birth, low birth weight, decreased APGAR scores at birth,

admission to special-care nurseries, reduced postnatal weight gain, persistent pulmonary

hypertension and poor neonatal adaptation (Chambers et al., 1996; Chambers et al., 1999;

Lattimore et al., 2005; Oberlander et al., 2006). Human studies have found that prenatal SSRI

exposure results in blunted infant heart rate and facial responses to a painful stimulus (heel

prick) at 2 days and 2 months after birth (Oberlander et al., 2002; Oberlander et al., 2005)

and altered hypothalamic-pituitary-adrenal (HPA) axis function at 3 months of age

(Oberlander et al., 2008).

The most commonly reported consequence of prenatal SSRI exposure is poor

neonatal adaptation (Laine et al., 2003; Oberlander et al., 2004; Sanz et al., 2005; Simon et

al., 2002; Zeskind & Stephens, 2004). This is a self-limited phenomenon usually lasting a

40

few days and comprising respiratory difficulty, cyanosis on feeding and jitteriness. There are

several possible mechanisms for these effects. One is an SSRI-elicited alteration in fetal brain

development and/or maturation. This is suggested by the results from our previous studies,

which involved 8-day administration of fluoxetine to chronically instrumented pregnant ewes

in late gestation. The fetuses of these ewes exhibited acute changes in fetal cardio-respiratory

function (Morrison et al., 2002), behavior (Morrison et al., 2001) and in HPA axis function

(Morrison et al., 2004). In addition, there was a transient reduction of postnatal weight gain,

similar to the human findings. The altered fetal behavior comprised a reduction in REM sleep

and increase in quiet sleep, while the altered HPA axis function comprised an increase in the

fetal prepartum cortisol surge and an apparent increase in the sensitivity of the adrenal gland

to ACTH. These prenatal alterations could thus contribute to the poor neonatal adaptation, as

well as some of the other adverse postnatal findings associated with prenatal SSRI exposure

in the human. However, other explanations for the poor neonatal adaptation have been

proposed. These include an SSRI withdrawal phenomenon in the postnatal period or a

persistence of drug in the newborn, leading to drug-related toxicity, i.e., the serotonin

syndrome (Andrade et al., 2006; Hendrick et al., 2001; Isbister et al., 2001; Koren, 2004;

Ruchkin & Martin, 2005). Moreover, FX and SSRIs are also used in children and adolescents

for treatment of depression and there are raising concerns about the safety of this therapy

(Sharp & Hellings, 2006; Usala et al., 2008). Therefore, we suggest that fluoxetine injection

to the newborn lambs and later in life at different ages is a useful model to test the hypothesis

which cannot be implemented in human because of ethical issues. In addition, as the lambs

were to be studied from birth to one year of age, longitudinal studies on cardiovascular,

41

metabolic and behavioral, and endocrine functions could be studied in relation to age, sex

and FX exposure.

1.9 Objectives:

To determine the ontogenetic changes in various physiologic, behavioral and

endocrine functions in lambs from birth to 1 year of age in relation to sex and acute postnatal

fluoxetine administration. The study involved 2 groups of lambs: one group received a bolus

FX injection at approximately 2, 10, 30 days and at about 3, 6 and 12 months of age and a

control group that was studied at the same time periods. In addition, postnatal outcomes in

the prenatally FX-exposed lambs were investigated with 2 groups of lambs: one group

maternally exposed to FX for 14 days of late gestation and one control group with no

prenatally FX exposure.

Specific objectives:

1. To examine the behavioral functions in newborn and postnatal lambs.

2. To examine cardiovascular function including blood pressure, heart rate and heart

rate variability in newborn and postnatal lambs.

3. To determine blood gas, oxygen saturation, pH, glucose, lactate in newborn and

postnatal lambs.

4. To investigate HPA axis function by measuring plasma cortisol and ACTH

concentrations in newborn and postnatal lambs.

42

5. To determine the effects of acute fluoxetine administration on the above variables

at 2, 10 and 30 days and 3, 6 and 12 months of age.

6. To investigate the postnatal outcomes in the lambs antenatally exposed to FX in

late gestation.

1.10 Hypotheses:

1. The major postnatal changes in behavior, cardiovascular and HPA axis

functions in postnatal lambs will be temporally related to weaning.

2. FX injection will cause transiently agitation in the newborn and postnatal

lambs, disrupt their feeding activities and thus affect their ultradian and circadian rhythms.

3. FX injection in the newborn lambs will transiently decrease their heart rate

and increase their arterial pressure, changing their heart rate variability.

4. FX injection will cause transient hypoxemia with the decrease in blood

oxygen saturation and Po2.

5. FX injection will alter the activity of HPA axis and lead to an increase in

cortisol and ACTH plasma levels.

6. Antenatally FX-exposed lambs will have adverse outcomes: lower birth

weight, and poor neonatal adaptation.

43

2. Materials and Methods

All procedures conducted on the animals were approved by the UBC Committee on

Animal Care and conformed to the guidelines of the Canadian Council on Animal Care. This

chapter describes the general materials and methods for Chapter 3 to 5

2.1.1 Animals:

Sheep were bred following a protocol in our lab to provide us with lambs for studying

all year round. The estrous cycle was synchronized via progestin containing vaginal pessaries

(Pharmplex, Milton NSW, Australia) for 14 days. Ovulation was then induced with an

intramuscular injection of pregnant mare serum gonadotropin (PMSG) (Wyeth, Ontario,

Canada). Ewes were then placed with a ram for natural mating. Subsequently, conception

was confirmed by measurement of plasma progesterone by the Gynecologic Endocrinology

laboratory in the Division of Reproductive Endocrinology and Infertility, UBC at 19 days

after pessary removal and gonadotropin injection. An ultrasound was performed at ~90 days

gestation to confirm a successful pregnancy. Pregnant ewes (Dorset/Suffolk) were

transported to the Child & Family Research Institute from Three Gates Farm, Gabriola

Island, at least 3 weeks before their due date (term = 147 days) to allow them to acclimate

with the new environment before their deliveries. They were housed singly in 2.4 x 1.2 m

pens or in groups of 2 in 2.4 x 2.4 m pens. Sheep were kept under a 12 hour light: 12 hour

dark cycle with scheduled lights on at 6:00 am and off at 6:00pm. The feeding regimen

comprised 0.6 kg grain and 0.7 kg local hay at 0730 am and 0.7 kg local and 0.7 kg alfalfa

hay at ~1430 pm. The grain ration was increased to 0.8 kg after delivery. Water was available

ad lib. Signs of imminent parturition in the ewe (bilateral concave flanks, restlessness,

44

pawing at the pen floor, moving in circles and looking toward the back) and the lambs’ birth

were revealed by digital video recording (DVR) from infrared cameras which will be

described in more detail in Section 2.2.5 (DVR and behavioral evaluation). Ewes were

allowed to deliver without assistance except in cases of malposition of the fetus. After the

lambs were born, they were dried with a towel, sexed, weighed and had an actiwatch placed

around their neck (more details about actiwatch in Section 2.2.6: Actiwatch/Rest activity

monitoring). Surgeries were conducted on the lambs on the first day after birth. Lambs were

housed in the same pen with their mothers until the weaning period. The study involved 2

groups of lambs: one group received a bolus fluoxetine injection at ~ 2, 10, 30 days and at ~

3, 6 and 12 months of age and a control group that was studied at the same time periods.

2.1.2 Surgical Preparation:

Surgeries were done on the lambs on postnatal day 1.5 ± 0.1 (for those who started

the day 3 experiment) or on postnatal day 9 ± 1.1 (for those who started the approximately

day 10 experiment). Lamb was anesthetized with 4% isoflurane and 40% NO delivered via a

face mask and then intubated with a cuffed endotracheal tube (3.5 to 5.0 mm O.D, depending

on the size of the lamb). Anesthesia was maintained with 1.5 to 2% isoflurane, 40% NO in

oxygen. The lambs were ventilated at a rate of with a frequency and tidal volume appropriate

for their weight and postnatal age (Moss et al., 1995). The eyes were lubricated with Refresh

Eye Lube. Bupivacaine hydrochloride (0.5%) was infiltrated subcutaneously at the base of

the ear before an ear tag was applied and at the surgical sites. Meloxicam (0.2 mg/kg) was

also given subcutaneously for analgesia and 250 mg ampicillin intramuscularly for antibiotic

prophylaxis. The lamb was placed on a heating pad during the surgery and a thermistor rectal

probe connected to a Yellow Springs Instruments Tele-Thermometer was used to monitor the

45

body temperature continuously. Heart rate was monitored intermittently via auscultation or

later in the study with a pulse oximeter (Nonin Medical Inc, Plymouth, MN, USA). Aseptic

surgical techniques were employed. All surgical instruments were autoclaved before surgery.

The skin area to be operated on the neck was shaved and washed with antiseptic soap, then

sprayed with 70% alcohol and Proviodine solution (USP 10%) with the combination repeated

3 times. A sterile surgical drape was used to cover the lamb with the surgical opening

positioned appropriately.

Access to the carotid artery and jugular vein was gained via an oblique transverse

skin incision in the neck from the sternomastoid muscle (from ~ 1cm lateral to just above the

jugular vein). Heparin-bonded 3.5 or 5 French polyurethane catheters (Instech Solomon,

Plymouth Meeting, PA) were filled with sterile heparinized 0.9% NaCl solution (12U

heparin/ml) and were implanted into the right carotid artery and jugular vein. The catheters

were secured in place with 2-0 silk non-reabsorbable sutures and then the catheters were

tunneled subcutaneously and exteriorized through a small skin incision at the dorsal neck.

These catheters were available for drug administration and blood sampling in the subsequent

series of experiments, while the arterial catheter was employed to measure arterial pressure.

The transverse and dorsal neck skin incisions were closed with 2-0 Polysorb reabsorbable

(Syneture, Canada) interrupted sutures. The skin and the outer surfaces of the catheters were

then cleaned with 70% alcohol. Then the distal ends of the catheters were sealed with sterile,

metal pins and the catheters were placed in a small zip-lock bag. The skin incisions were

covered with sterile gauze pads, over which was placed in a zip lock bag into which the

catheters were placed. An elastic bandage was then placed loosely around the neck and

secured with a safety pin.

46

To prevent the risk of fly strike when the lamb gets older, all newborn lambs also had

their tails docked. The section of the tail to be cut was first shaved, disinfected, infiltrated

with 0.5% bupivicaine and then cut and cauterized using a Supervet Tail Dock

Cutter/Cauterizing Instrument (Syrvet Inc., Iowa, USA). Following this, the skin margins of

the tail stump were opposed using a continuous 2-0 Polysorb suture.

Lambs were kept in the operating room until they could breathe spontaneously and

were conscious. They were then returned to their mother. The total surgery time from

removing the lamb from its mother to returning the lamb post-surgery averaged 2.2±0.01 h.

The lambs were weighed daily in the morning. Catheters were flushed daily with sterile

heparinized 0.9% NaCl solution (12U heparin/ml) to maintain their functioning until the last

experiment (1 year of age). If catheters stopped functioning, another surgery was performed

to implant other appropriate catheters at least 2 days before the experiments were started.

2.1.3 Experimental Protocol:

The experiments on the lambs began on the day following surgery. A sterile silicone

urinary catheter was placed in the urethra of the female lambs and secured with tissue

adhesive and a 2-0 silk suture tied to the wool in the groin area on the day before FX

experiments. Since in male lambs, the urethra makes a hairpin turn, it is impossible to insert a

urethral catheter. A strap-like urine collection device, which was successfully developed by

Tim Chow, another PhD candidate in our lab, was placed on the lamb’s penis, wrapped

around the abdomen and tied securely on their back about 30 minutes before the FX

experiments. This served for urine collection mainly for Tim Chow’s project which involves

developmental fluoxetine pharmacokinetics and metabolism. The urine volume data was also

47

used in chapter 3 to determine the development of urine flow rate in the lambs with

advancing age. For the experiments on the 2, 10 and 30 day old lambs, two experiments were

carried out on the same day, starting at 8:30am for the first lamb and 11:30am for the second.

The lambs were placed in a cloth sling set up in the pen that contained the lamb’s mother or

the biggest pen. The lambs were suspended above the floor in the sling and normally lay

quietly or slept. They were only in the sling for the first two and a half hours of the

experiment, and then were returned to their mothers, so as not to disrupt the normal suckling

regimen. Lambs at 10 days and 1 month of age were bleating and jumping sometimes

especially toward the end of the 2.5h duration. For the experiments conducted at 3 months

and beyond, the lambs were placed in a small monitoring pen (120 X 65 cm) set up in one

corner of the holding pen. They remained in this pen for the duration of each experiment

(24h for the control experiment and 72h for the FX experiment) and had access to food and

water. Since lambs started having solid food around 2 months old, experiments on lamb at 3

months and above were started at 9:00am (30 min after lambs finished their feeding) to

eliminate the affect of eating on changes in heart rate and blood pressure (Jones, Langille,

Frise, & Adamson, 1993).

Each experiment involved i.v administration of sterile water (0.1ml/kg, equal to the

volume of 10mg/ml FX given in FX experiment) or fluoxetine (1mg/kg) followed by serial

collection of arterial blood samples (~ 3 ml) at -30, -15, 5, 15, 30, 45 and 60 minutes and 2,

4, 6, 9, 12, 24, 36, 48, 60 and 72 h (more specifics in table 2). The total volume of blood

collected (42ml) represents ~ 8% of the total blood volume in the newborn lamb and much

less in the older lambs.

48

FX Blood gases ACTH, cortisol -30min -15min SW/FX i.v 5min 15min 30min 60min 2h 4h 6h 9h 12h 24h 36h 48h 60h 72h

Table 2.1: Time points at which blood samples were collected for arterial blood gases, FX, ACTH and cortisol measurement. SW = sterile water.

The symbols indicate when the samples were taken for the corresponding variables

2.1.4 Sample Collection:

Blood samples for hormone and fluoxetine measurements were collected through the

carotid artery catheter and placed in a 4ml EDTA K2 and Lithium Heparin (68USP units)

vacutainer tubes (BD Vacutainer, NJ, USA) and centrifuged for 20 min at 4000 rpm in a

Centrifuge 5810R (Eppendorf, ON, Canada). Plasma was then transferred to polyvinyl

Eppendrorf tubes for hormone samples and glass tubes for fluoxetine samples and stored at -

20oC until analysis. Arterial pressure was obtained from the arterial catheter using a

disposable pressure transducer and Powerlab data acquisition system, which also calculated a

heart rate from the pulse pressure. The ECG was recorded using surface neonatal ECG

49

electrodes and the Powerlab Heart Rate Variability Module was used to assess heart rate

variability (HRV).

Blood samples were also collected before the surgery, on postnatal day 2.3 ± 0.2 (n =

11), for ACTH and cortisol measurements. Lambs were taken to the operating room and their

wool along the jugular vein was shaved. The skin was disinfected with alcohol 70% and

applied with local anesthetic Xylocain 2%. Approximately 2ml of blood was taken to the

EDTA K2 and Lithium Heparin (68USP units) vacutainer tubes via venipuncture. Lambs

usually slept while blood was collected and were returned to their mother immediately.

Samples were centrifuged and plasma was transferred and stored as mentioned above.

2.1.5 Digital Video Recording And Behavioral Evaluation:

One infrared video camera were installed on the wall of each pen in the sheep holding

room and connected to a digital video recording system on a computer in the lab, which was

directly adjacent to the sheep room. Another mobile infrared camera was put outside the pen

or above the monitoring pen to allow for the recording and observations of the lambs from a

direct view, as the lambs sometimes could not be observed by the fixed cameras because of

the ewe and lamb’s position within the pen. On the day of experiment the mobile camera was

also be used to record behavioral changes immediately after FX administration. The infrared

function allowed the recording and monitoring the ewe and lamb’s behavior at night without

interrupting their circadian rhythm. These cameras could also be accessed remotely from

home and the video records could be analyzed off-line.

Lamb’s activities were categorized into lying down sleep, lying down awake,

standing (still or moving), walking, suckling attempt, successful suckling and playing.

50

Playing activities included running, jumping, climbing on their mother’s back, nudging their

mother, nosing and playing with hay. These activities were collected for the dominant

activity in 15 second epochs. The collected activities from the DVR were then matched with

the actiwatch data (see below) to obtain an equivalent score from the actiwatch record for the

activity identified from DVR in the same epoch. These scores were used to determine the

intensity of each kind of activity. After each type of activity was identified as mentioned

above from DVR observations, only standing activity was further analyzed using a cut-off

point of 60 on actiwatch score to separate standing still from standing moving. In other

words, standing still was defined as standing activity from DVR observation and

corresponding actiwatch score lower than 60. Similarly, standing mobile was standing

activity from DVR and corresponding actiwatch score equal or greater than 60. The

percentage of time per 24 hour lambs spent for each activity was also calculated.

2.1.6 Actiwatch Rest/Activity Monitoring:

An Actiwatch Activity Monitor (AW64, Mini Mitter Inc., Bend, OR, USA), which is

a small wrist watch sized device, was enclosed in a protective metal case and placed around

the neck of the lamb with a dog collar from the first day after birth until at least 90 days after

birth to measure rest-activity cycles and sleep patterns. The actiwatch contains an

omnidirectional accelerometer which detects and stores the occurrence and intensity of any

movement with a force of at least 0.01g. The activity data was stored in 15s epochs and

downloaded every 10 days using an Actiwatch reader and analyzed using an Actiware-Sleep

version 3.5 software (Mini Mitter). The analyzed variables included total daily activity score

(total number of movement counts in each 24h period), active bouts/day (= mean activity

score, i.e magnitude of activity per epoch), mean movement score in active (the average

51

number of movement counts during bouts of activity), episode duration (= moving minutes/

mean activity score), % time moving and % time immobile.

The ultradian and circadian rhythms in the lambs were determined using Fast Fourier

Transformation (FFT) analysis from the Actiware software. The four most prominent

frequencies in the FFT analysis were selected.

2.1.7 Powerlab Data Acquisition System:

The lamb’s carotid artery catheter was connected to a disposable pressure transducer

(DTXTM Plus TNF-R; Becton Dickinson, Singapore), which was connected to a polygraph

recorder (model TA-4000, Gould Instrument Systems Inc, Valley View, OH) and to a

Powerlab data acquisition system (AD instrument, Mountain View, CA, USA) to

continuously record heart rate and arterial pressure throughout the first 2h of the experiments

in 4, 10 and 30 day old lambs and for the entire 24h or 72h of the experiments at 3, 6 and 12

month old lambs. The transducer was kept on the platform which was adjusted to the lambs’

heart level when they stood or were in the sling. Three ECG snap electrodes were attached to

the skin on the back and bilaterally on the chest and secured in place by adhesive bandages.

During the experiments these electrodes were connected to the Powerlab system by three

snap leads to record the electrocardiogram. The Powerlab Heart Rate Variability Module was

used to assess and analyze heart rate variability (HRV) including estimates of

parasympathetic and sympathetic tone.

52

2.1.8 Arterial Blood Gases Analysis:

Blood samples were collected at -15, 5, 15, 30, 60 and 120 minutes to measure acid-

base balance (pH), base excess (BE), bicarbonate (HCO3-), arterial blood gases (pO2, pCO2),

electrolyte (Na+, K+ and iCa2+) using an I-STAT-1 analyzer (Abbott, ON, Canada) and CG8+

cartridges (Abaxis, Union City, CA, USA). Body temperature was measured continuously

with the thermistor rectal probe (YSI Instruments, Yellow Springs, OH) over the first 2h of

experiment or later by a regular digital thermometer and blood gas results were corrected

with the appropriate temperature when the sample was collected. Hemoglobin concentration

and oxygen saturation were measured with a Radiometer OSM3 Hemoximeter (Radiometer,

Copenhagen, Denmark). Glucose and lactate were determined with a YSI glucose-lactate

analyzer (YSI Inc., Yellow Springs, OH).

2.1.9 Adrenocorticotropin Hormone Assay:

ACTH level was measured using a commercially available human ACTH ELISA kit

(Calbiotech Inc., CA, USA) that detects ACTH1-24. The kit has previously been validated for

use in sheep (You et al., 2008). 200µl of plasma was added together with 25 µl of

biotinylated antibody and 25µl of enzyme labeled antibody into each well of the microplate.

The microplate then was covered with aluminum foil to prevent light exposure and was

shaken at 170 ±10 rpm for 4.5 hour at room temperature. After the remaining antibodies were

washed with wash solution five times, 150 µl of TMB substrate was added into each well and

the plates shaken at the above speed for 30 minutes at room temperature. 100 µl of stopping

solution was then added to terminate the reaction. ACTH level was determined by an ELISA

reader (initially by Dynex Technologies and later by Wallac Victor3V 1420) set at 450nm.

53

Each sample was measured in duplicate. The sensitivity of the assay was < 1 pg/ml. The

intra- and interassay coefficients of variation were 7.27% and 7.08%, respectively.

2.1.10 Cortisol Assay:

Commercially available sheep cortisol ELISA kits (Calbiotech Inc., USA) was used

to determine cortisol concentrations. 40µl of sample in duplicate and 200µl of Cortisol

enzyme conjugate were added into each well coated with anti-cortisol monoclonal antibody

for competitive binding. The microplate was then incubated for 1 hour at room temperature

with shaking. The unbound hormone and conjugate was washed off with washing solution 3

times. 100µl of TBM substrate was added and incubated for 15 minutes in the same previous

condition. The reaction was brought to end by adding 50 µl of stop solution in each well. The

cortisol level was determined by ELISA reader (initially by Dynex Technologies and later by

Wallac Victor3V 1420) at 450nm. The sensitivity of the assay was 1.38 ng/ml. The intra- and

interassay coefficients of variation for low control (78 ± 3 ng/ml) were 10.7% and 3.2%, and

for high control (225 ± 5 ng/ml) were 7.2% and 6.7%, respectively.

2.1.11 FX and NFX assay:

FX and NFX concentrations were measured by Dr.Tim Chow from Dr. Wayne Riggs’ lab,

Faculty of Pharmaceutical Science, UBC using a validated liquid chromatography with

tandem mass spectrometry method (LC/MS/MS) (Chow et al., 2011). The sample volume

used for each enantiomer (R)-, (S)- fluoxetine and (R)-, (S)- norfluoxetine was 250 µl. The

limit of quantitation was 1 ng/ml.

54

2.1.12 Statistical Analysis:

All data were analyzed using the Sigma Plot 11 statistics software (Systat Software,

Inc; San Jose, CA, USA). A 2 way General Linear Model (GLM) for repeated measures

ANOVA or a three way ANOVA followed by the Bonferroni t-test for multiple comparisons

was performed to determine the effects of age, sex and FX treatment on the interested

variables. P < 0.05 defines significant differences. Data are presented as mean ± SEM. Other

statistical methods that were used in particular results chapters are described in those

chapters.

55

3. Postnatal Lamb Development and Sex Differences

3.1 Introduction:

The geometry of the circulation in the newborn alters remarkably at birth, due to the

loss of the umbilical circulation, onset of pulmonary ventilation and increase in pulmonary

blood flow. With the onset of air breathing, the pulmonary vascular resistance decreases and

the surface area for gas exchange increases. Hemodynamic changes result in the closure of

fetal circulatory shunts – formamen ovale and ductus arteriosus. At birth, cardiovascular

function also alters significantly with an increase in arterial pressure, heart rate and cardiac

output. Much research has been done regarding the cardiovascular and hemodynamic

changes immediately following birth. (Klopfenstein & Rudolph, 1978; Lister et al., 1979;

Minoura & Gilbert, 1987; Woods et al., 1977) However, little has been known about these

physiological developments (heart rate, blood pressure, HRV) in lambs after the initial

postnatal period. In lambs, heart rate and blood pressure increase with feeding (Jones et al.,

1993) but whether there is any effect of weaning on the development of these variables is

unknown. Studies in goats found an elevated cortisol and B-endorphin concentration during

suckling in kids but no significant changes in heart rate or blood pressure related to suckling

(von Walter et al., 2010).

Heart rate variability (HRV) represents the oscillation of the intervals between

consecutive heart beats. It is in part determined by influences of the autonomic nervous

system on the heart. Since HRV is a non-invasive method to measure the autonomic

impulses, monitoring HRV variables are used clinically as an evaluation of fetal well being

in the human fetus (Alfirevic et al., 2006; Graatsma et al., 2009; Maso et al., 2012; Van

56

Leeuwen et al., 1999) and also in the neonate in neonatal intensive care (Fairchild & O'Shea,

2010). They are also used in a number of conditions in the adult, including coronary artery

disease, systemic hypertension and myocardial infarction (Charkoudian & Rabbitts, 2009;

Huikuri, 1995; Pagani & Lucini, 2001). However, there is less information about changes in

HRV characteristics during development, including the changes which occur around

weaning. Even though high HRV usually indicates a good adaptation of an effective

autonomic system in a healthy individual while low HRV often indicates the presence of

physiological malformation, the threshold is age-dependent. HRV has been shown to be

lower in healthy women than in men (Saleem et al., 2012); however, it is not known if the

same situation exists in sheep. There are established data on the differences in the prevalence,

characteristics and response to treatment of cardiovascular diseases (Dunlay & Roger, 2012).

After adjustments for the increased longevity in women, age-specific stroke incidence is

generally higher in men than women (Katsiki et al., 2011). Gender differences in cardiac

autonomic control might occur early in life and could be one of the potential mechanisms for

the observed differences in cardiovascular disease between males and females.

Birth also results in changes in the surrounding environment. The newborn has to

adapt with and become entrained to the circadian rhythm of the light dark cycle. Melatonin,

which is secreted from the suprachiasmatic nucleus under the influence of light from the

environment, is not present in human infants until 9-15 weeks of age (Kennaway et al., 1996)

and in lambs at 3-4 weeks after birth (Kennaway et al., 1996; Nowak et al., 1990). A

disrupted circadian rhythm may play a role in the pathogenesis of certain illnesses such as

coronary heart disease, breast cancer, gastrointestinal disturbances, sudden infant death

syndrome even though the mechanisms involved have not yet been established (Mosendane

57

et al., 2008; Lohr & Siegmund, 1999). In a previous study using actigraphy in newborn

lambs, our lab found a short (~ 1 h) activity rhythm not present in adult sheep, in which the

circadian rhythm was the most dominant. Only the lactating ewes had a short (~1 h) rhythm

as the second, third or fourth most dominant rhythms. Also, the total daily activity level in

the lambs over the first ~3 weeks was significantly higher than in adult sheep (Rurak et al.,

2008). However, it is still not yet clear when the short term ultradian rhythm in the lamb

disappears and when the circadian rhythm becomes dominant as in the adult sheep. In the

human, babies who are breast-fed for the first 2 years of life wake up more often at night and

have shorter sleep bouts instead of having a long unbroken night sleep (Elias et al., 1986).

Thus the short 1 h rhythm observed in the lambs may be the result of suckling activity.

3.2 Methods:

3.2.1 Experimental Protocol:

As described in chapter 2, the lambs in this study were used in a longitudinal study of

the disposition of the antidepressant, fluoxetine (FX), from birth to one year of age. All the

data used in this chapter was from the values of control experiments with sterile water

injection or from the control time point of FX experiments (i.e: 30 mins before FX injection)

except for the urine volume data and rest-activity cycles. The animal housing, surgical,

experimental and analytical methods are described in Chapter 2.

3.2.2 Statistical Analysis:

A 2 way General Linear Model (GLM) for repeated measures ANOVA followed by

the Bonferroni t-test was used to determine the effect of age and sex in cardiovascular,

58

metabolic and endocrine functions. The relationship between postnatal age and the

cardiovascular, metabolic, endocrine and urinary variable was analyzed using piecewise

linear regression with 2 elements. The area under the curve (AUC) of the activity variables

(total activity score, mean activity score, mean score in active, episode duration, % time

mobile, % time immobile, circadian and ultradian rhythms) was calculated for each lamb and

analyzed by 2 way ANOVA to determine the effect of age and gender in their rest-activity

cycle.

3.3 Results:

For the newborn lambs, the average age of the lambs on the control and FX

experiments is 3.2 ± 0.5 and 4 ± 0.4 postnatal days, respectively

3.3.1 The Development Of Physiological Variables And Sex Differences In

Development From The Newborn Period Up To 1 Year Of Age:

The lambs’ heart rate fell dramatically over the first month of life from 220.4 ± 6.3

bpm on postnatal day 3 to 155.2 ± 8 bpm at 1 month of age. A further decrease in heart rate

occurred thereafter to 94 ± 3.4bpm at 1 year of age. There was a significant difference in the

values between 10 days, 1 month, 3 months and 6 months. There was also a sex difference in

heart rate development. At 6 months, heart rate in female lambs was significantly lower than

male lambs (96 ± 7.8 bpm in female vs 115 ± 7.7 bpm in male, (p = 0.016) while the

situation seemed to be reversed at 1 month of age (167 ± 10.5 bpm in female and 155 ± 8bpm

in male) even though the difference was not significant at 1 month. However, they both

reached a similar heart rate at 1 year of age (97 ± 3.9 bpm in female and 93 ± 5.2bpm in

male). Female lambs tended to reach the adult heart rate value earlier than males, at 6 months

59

of age. Arterial pressure development was similar in male and female lambs. Arterial

pressure gradually increased from 66 ± 1.5 mmHg on day 3 to 82.9 ± 1.1 mmHg at 1 year of

age and the value was significantly different at 1 month and 1 year-old against 3 days of age.

(Fig. 3.1)

In terms of heart rate variability, a sex difference was only found in Low Frequency

(LF) power. At 3 day-old, LF power in male was significantly higher than that in female

(52.4 ± 6.2 nu in male vs 25.7 ± 6.5 nu in female, p = 0.036). In female, LF power increased

to peak at 10 day-old (61.5 ± 5 nu) and then decreased to a plateau from 3 months to 1 year

of age (range: 38 ± 6.8 – 43.4 ± 4.9nu). In males, LF power decreased from 52.4 ± 6.2 nu on

day 4 to a plateau between 4 and 12 weeks (range: 35.8 ± 3.8 – 46.9 ± 5.9 nu ), then

increased at 6 months (56.1 ± 4.2 nu), followed by a decrease at 1 year of age (Fig. 3.3A).

The time domain variables of HRV (including SDNN, SD Delta NN, RMSSD) were

significantly different between the 3-day, 10 day-old and the remaining ages (1, 3, 6 months

and 1 year old) with the lower values at 3 days of age (22 ± 1.7 msec for SDNN, 19.4 ± 3.4

for SD Delta NN and RMSSD) and higher values at 1 year of age (102.8 ± 12.4 for SDNN,

80.5 ± 9.8 for SD Delta NN and RMSSD) (Fig. 3.2). High Frequency (HF) power tended to

increase from 18.4 ± 2 nu on day 3 to 28.3 ± 2.2 nu at 1 year of age with the lowest value at

10 days of age (16.4 ± 1 nu). Since HF power did not change much over the time, changes in

LF/HF ratio were due mainly to LF power alterations. The LF/ HF ratio peaked at day 10

(3.7 ± 0.48) and was significantly higher than that at 3 months (1.8 ± 0.3) and 1 year of age

(1.6 ± 0.2). (Fig. 3.3)

Fig. 3.4 show an inverse relationship between SDNN (msec) and heart rate in the

lambs from 3 days to 1 year of age with r2 = 0.55, p < 0.001

60

FIG. 3.1: Postnatal lamb arterial pressure (A) and heart rate (B) in all (open bar), male (black bar) and female (gray bar) lambs at 3 days (n = 14), 10 days (n = 18), 1 month (n = 15), 3 months (n = 17), 6 months (n = 11) and 1 year (n = 10) of age. Different letters (a,b,c and d) indicate significant differences between ages. Asterisk indicates significant difference between male and female, (p < 0.05). 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

61

FIG. 3.2: Time domain of heart rate variability: SDNN (standard deviation of NN interval) (A), SD Delta NN (Standard Deviation of the differences between adjacent NN intervals) (B), RMSSD (Square root of the mean of the squared differences between adjacent NN intervals) (C) in lambs at 3 days (n = 11), 10 days (n = 18), 1 month (n = 15), 3 months (n = 17), 6 months ( n = 11) and 1 year ( n = 10) of age. Different letters (a,b, and c) indicate significant differences between ages, (p < 0.05). 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

62

FIG. 3.3: Frequency domain of heart rate variability: LF (Low Frequency) power (A), HF (High Frequency) power (B), LF/HF ratio (Low Frequency/High Frequency Ratio) (C) in lambs at 3 days (n = 11, male n = 4, female n = 7 ); 10 days (n= 18, male n = 9, female n = 9), 1 month (n= 15, male n = 8, female n = 7); 3 months (n= 17, male n = 9, female n = 8), 6 months (n = 11, male n = 6, female n = 5), 1 year (n = 10, male n = 6, female n = 4) of age. Different letters (a,b) indicate significant differences between ages. Asterisk indicates significant difference between male and female, (p < 0.05). 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

63

FIG. 3.4: The relationship between SDNN (msec) and heart rate (bpm) in the lambs from 3 day to 1 year of age

Among the blood chemistry variables collected (glucose, lactate, O2 sat, pO2, pCO2,

pH, BE, HCO3, Hb), only the pH value at day 3 was different between male and female (Fig.

3.5), with the value in males (7.379 ± 0.0016) being significantly lower than in females

(7.457 ± 0.018), p < 0.0001). Following birth, Po2 and pH increased from 86.6 ± 3.4 mmHg

and 7.421 ± 0.016 on postnatal day 3 to 110.7 ± 3.27mmHg and 7.489 ± 0.009 at 1 year of

age, while Pco2 decreased from 41.9 ± 1.01 mmHg on postnatal day 3 to 35.57 ±

0.653mmHg at 1 year of age, with the main changes occurring over the first 12 weeks. Base

excess tended to increase from 3 ± 1.019 mmol/l on postnatal day 3 to peak 5.647 ± 0.342

mmol/l at 3 months of age and slightly decrease thereafter. Both glucose and lactate

concentrations decreased from 5.39 ± 0.215 mmol/l and 1.028 ± 0.055 mmol/l on postnatal

day 3 to 2.91 ± 0.12 mmol/l and 0.527 ± 0.042 mmol/l at 1 year of age, respectively, again

with most of the change occurring in the first 12 weeks, although lactate decreased further at

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6 months. Hemoglobin concentration decreased more rapidly, from 12.2 ± 0.34 g/l at 3 days

of age to low value 8.729 ± 0.418 g/l at week 4, and was relatively constant thereafter

(Fig.3.6-3.8). These suggested that the newborn lambs appeared to be more hypoxemic and

acidemic than the adult.

Urine flow rate (ml/min.kg) was based on the mean of urine flow rate at different

intervals of the FX experiment and was normalized for the weight of each lamb at the

corresponding age. Urine flow rates at 2 and 10 days of age were significantly higher than

the values at 3, 6 and 12 months of age. There was no sex difference in urine flow rate at any

age. (Fig. 3.9)

The cortisol concentration was not significantly different between day 3 (day of

control experiment) and ~ day 2.3 ± 0.2 before the surgery to implant the catheters for the

subsequent experimental sampling.

In fact, combining the mean cortisol values in the fetus lamb in late gestation from a

previous paper in our lab (Morrison et al., 2004) suggested the lambs’ prior surgery cortisol

value was consistent with the cortisol surge which usually occurs right before birth in the

sheep. After the day 3 experiment, cortisol concentrations at 10 days and 1 month gradually

decreased, but are significantly higher than the values at 3 and 6 months. At 3 months, the

cortisol concentration became stable through to 1 year of age. (Fig. 3.10) There were no

statistical significant changes in ACTH concentration from before surgery up to 1 year of

age. (Fig. 3.11)

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FIG. 3.5: Mean pH value in total (open bar), male (black bar) and female (gray bar) lambs at 3 days (n = 13, male n = 6, female n = 7 ); 10 days (n= 17, male n = 8, female n = 9), 1 month (n= 15, male n = 8, female n = 7); 3 months (n= 17, male n = 9, female n = 8), 6 months (n = 10, male n = 5, female n = 5), 1 year (n = 10, male n = 6, female n = 4) of age. Different letters (a,b,c) indicate significant differences between ages. Asterisk indicates significant difference between male and female, (p < 0.05). The fetal value at 147 days of gestational age (at term) is taken from Rurak & Bessette, (2012) and was not included in the statistical test. 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

66

FIG. 3.6: Mean pCO2 (A), pO2 (B) and O2 sat (C) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. Different letters (a, b, c, d) indicate significant differences between ages, (p < 0.05). The Fetal values at 147 days of gestational age (at term) are taken from Rurak & Bessette, 2012 and was not included in the statistical test. 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

67

FIG. 3.7: Mean HCO3 (A), Base Excess (B) and Hemoglobin (C) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. Different letters (a,b)indicate significant differences between ages, (p < 0.05). The Fetal values at 147 days of gestational age (at term) are taken from Rurak & Bessette, 2012 and was not included in the statistical test 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

68

FIG. 3.8: Mean Glucose (A) and Lactate (B) in lambs at 3 days (n = 13); 10 days (n= 17), 1 month (n= 15); 3 months (n= 17); 6 months (n = 10) and 1 year (n = 10) of age. Different letters (a ≠ b ≠ c≠ d) indicate significant differences between ages, (p < 0.05). The Fetal values at 147 days of gestational age (at term) are taken from Rurak & Bessette, 2012 and was not included in the statistical test 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

69

FIG. 3.9: Mean urine flow rate in lambs at 3 days (n = 10); 10 days (n= 10), 1 month (n= 9); 3 months (n= 12); 6 months (n = 7) and 1 year (n = 10) of age. Different letters (a ≠ b) indicate significant differences between ages, (p < 0.05). Fetal value at 130-140 days of gestational age is for reference and was not included in the statistical test. The value is taken from (Turner et al., 2008) GA = gestational age, 3d = 3 days; 10d = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year

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FIG. 3.10: Mean cortisol concentration in fetal lambs from preinfusion to infusion day 2 and from infusion day 5 to 8 adapted from Morrison et al, 2004 and in postnatal lambs prior surgery (n = 11), at 3 days (n = 14); 10 days (n= 12), 1 month (n= 12); 3 months (n= 12); 6 months (n = 8) and 1 year (n = 8) of age. Different letters (a ≠ b ≠ c ≠ d ≠ e ≠ f) indicate significant differences between ages, (p < 0.05)

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FIG. 3.11: Mean ACTH concentration in fetal lambs from preinfusion to infusion day 2 and from infusion day 5 to 8 adapted from Morrison et al, 2004 and in postnatal lambs prior surgery (n = 11), at 3 days (n = 2); 10 days (n= 2), 1 month (n= 2); 3 months (n= 14); 6 months (n = 3) and 1 year (n = 4) of age.

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Cortisol, ACTH rhythm in the newborn and postnatal lambs:

At 3 day, 3, 6 month and 1 year–old experiment, the cortisol level at 30min, 1h and

2h tend to be higher than that at other time points during the day but the differences were not

statistically significant. However at 10 days and 1 month, respectively the higher cortisol

value at 30 min (at 10:00am) (201 ±64.9; 145.6 ± 24.8 ng/ml), 1 h (at 10:30am) (209 ±76.6;

178.1 ± 35.6ng/ml), and 2 h (at 11:30 am) (231.8 ± 72.5; 183.5 ± 46 ng/ml) were

significantly different from that at 6 h (at 15:30 pm) (85.7 ± 25.6; 68.2 ± 13 ng/ml) and 12 h

(at 21:30 pm) (78.1 ± 17.3; 73.4 ± 19.8 ng/ml), respectively. (Fig. 3.12)

From 3 month onward, lambs were put in the monitoring pen during the whole period

of the experiment (4 days). The cortisol level did not change much over the sampling time

point during the day with placebo injection at 3 month, 6 month and 1 year-old experiment,

but there was a sex difference in cortisol concentration at 30 min (10:00 am), 1 h (10:30 am),

6 h (15:30 pm) and 12 h (21:30 pm) samples when the lambs were 1 year old. Cortisol levels

were siginificantly higher in males than females at 1 year of age (p = <0.001).

3.3.2 Neonatal Behavioral Development:

From the DVR observations on 35 lambs following birth, we found the average times

that it took for the lambs to first stand, walk and attempt to suckle were 33 ± 3, 45 ± 4 and 57

± 5 minutes, respectively. Our results are similar to what have been shown in the literature

(Dwyer, 2003; Fraser & Broom, 1997). However, we did not see any effects of sex or lamb

number on the time that lambs took to first stand, walk or suckle. Also, we did not see any

significant correlation between these first time activities and birth weight.

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FIG.3.12: Cortisol concentration during the day in the lambs at 10 days, 1 month and 1 year of age. Samples were collected at -30, -15 min before sterile water injection and 30 min, 1,2,6 and 12 hour after the injection (10 days group, n= 9; 1 month group, n = 11; 1 year group, n = 8). Different letters (a ≠ b) indicates significant differences between the time points. Asterisk indicates significant differences between male and female

74

Activities on postnatal day 2-3 were observed from a smaller group of normal term

lambs (n= 11) who were not subjected for any experiments or surgery at this age. The results

showed lambs at this age spend most of the time at rest or asleep (~ 74.4%). Ewes of twin

and triplets appear more frequently to initiate suckling than their lambs at this age. In

general, only 33 ± 7% the number of bouts was initiated by the lamb itself. In terms of infant-

maternal bonding, we observed that lambs sleep close to either their mom or with their

siblings most of the time. Only one single female lamb showed less attachment to her

mother.

In addition, I compared the time spent on suckling on postnatal day 2-3 in single

(n=6), twin (n= 14) and triplet (n=3) lambs. The suckling time included both suckling

attempts and successful suckling. The triplets spent significantly more time suckling than the

singles and the twins. However, they gained less weight than the singleton and twins

although the differences were not significant (p = 0.064) (Fig. 3.13)

3.3.3 The Development Of Circadian And Ultradian Rhythms. The Ultradian 1 h

Rhythm Is Associated With Suckling:

Since during the period of the circadian rhythm study (i.e from birth to postnatal day

90), the lambs used in this study were also subjected to acute FX exposure intermittently at

approximately day 4, 10 and 30, it is important to note that the FX administration did not

result in any obvious physiological or behavioral changes in the lambs at day 4 and 10. Since

the activity was lower on FX day experiments in lambs at 1 month of age, those who were

subjected to the 1 month FX experiment were excluded from the regression analysis of this

circadian rhythm study. The results of FX administration are not included here.

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The daily total activity score up to postnatal day 90 of the rest activity cycle is shown

in Fig. 3.14. There were no differences in the rest activity cycle development in male and

female lambs (data not shown). The development of four most dominant FFT rhythms for 21

lambs for 90 days after birth are shown in Fig. 3.15. The results indicate that in the newborn

the 1-2 hour ultradian rhythm is the most dominant. About 3 weeks later, the ~ 1 hour rhythm

disappears and the circadian rhythm becomes dominant. Subsequently, an 8 hour ultradian

rhythm becomes secondarily dominant. A third rhythm, a 6 hour ultradian rhythm also

developed at ~ 20 postnatal day, and by about postnatal day 65, this rhythm was consistently

the third or fourth most dominant rhythm. No sex differences were found in circadian and

ultradian rhythm development (data not shown).

The 1-2 h rhythm in the lambs, which was not observed in the adult sheep,

disappeared around 3 weeks after birth. At the same time, the total activity also decreased

and became stable (Fig. 3.15). Regression analysis of the day that the 1h rhythm disappeared

against the day that the lambs had the lowest activity during the first 90 postnatal days

indicated that there was a significant correlation between the ~ 1-2 h rhythm and total

activity (r² = 0.5375, p < 0.0001) (Fig. 3.15). In addition, the correlation between sucking

frequency from DVR observations and that from FFT in 21 individual lambs on postnatal day

2 and 3 was also significant (r² = 0.1947, p = 0.045) (Fig. 3.17). This suggests that the 1-2 h

rhythm only seen in the lambs is associated with suckling.

The correlation between the first day the 6h rhythm appeared and the first day lambs

ate significant amounts of hay was also statistically significantly (r² = 0.4973, p = 0.00107)

which indicated the link between their rumen development and the 6h rhythm (Fig. 3.16).

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FIG. 3.13: Weight gain (A) and total suckling time (B) in singleton (n = 6), twin (n =14 for weight gain and n = 10 for total suckling time) and triplets (n = 3) in lambs on postnatal day 2-3 (N = 22). Asterisk indicates significant difference between singleton and triplets, (p < 0.05)

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FIG. 3.14: Plots of daily total activity score (a), activity bouts/day (b), mean score in active period (c), mean episode duration (d), % time moving (e) and % time immobile (f) against postnatal age in lambs. (n=21)

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FIG. 3.15: (A) The average development of 24, 8, 6 and 1-2 hour activity-rest rhythms. The lines with diamond, square, triangle and x symbols indicate the 24h circadian, the 8 hour, the 6 hour and 1 hour ultradian rhythms, respectively. Levels of rhythm dominance of 1 to 4 indicate first to fourth most dominant rhythms. A level of rhythm dominance of greater than 4 indicates that this rhythm is not one of the top four rhythms (n=22). (B) The daily mean total activity score. (n= 21)

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FIG. 3.16: (A). Relationship between the day that the 1h rhythm disappears and the day of the lowest total activity (n = 21). (B) Relationship between the day that the 6h rhythm disappears and the first day lamb eating hay (n = 21)

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FIG. 3.17: Relationship between suckling frequency on postnatal day 2-3 from DVR observation and calculated from the actiwatch FFT (n = 21)

The growth rate was significantly different between the 1st and 2nd of the 25-day

intervals and the 3rd, 4th of the 25-day intervals (p< 0.001). The slope of the daily weight

gain decreased from 279.0 ± 1.86 g/d for the first 25 days to 209.3 ± 3.03, 142.7 ± 1.95,

143.6 ±7.54 and 122.1 ± 13.2 g/d for the subsequent 25 day intervals.

3.3.4 Weaning And Changes In Physiological Variables:

In our study, the time of weaning was defined as a window between when lambs

started to first consume solid food (mostly hay) to when the 1h rhythm due to suckling

disappeared (i.e., from 29.6 ± 1.1 to 50.9 ± 4.8 days). We have used piecewise linear

regression with 2 elements to determine the break point which is the point where the slope of

the regression curve for the variable versus age plot changed abruptly. The break points of

the significant changes in heart rate, SDNN, glucose, pO2 and urine flow rate were at 41.1 ±

81

5.5 (p <0.0001); 51.6 ± 13.3 (p = 0.0002); 61.1 ± 7.9 (p <0.0001); 52.3 ± 22.3 (p = 0.0213)

and 38.4 ± 8.7 (p < 0.001) days of age, equivalent to 5.87 ± 0.78; 7.37 ± 1.9; 8.72 ± 1.12;

7.47 ± 3.18 and 5.48 ± 1.2 weeks of age, respectively. However, arterial pressure, LF/HF

ratio, Lactate, Hb and cortisol changed significantly with the break points at 16 ± 5 (p =

0.002); 5 ± 2 (p = 0.014); 6.5 ± 2.4 (p = 0.0084); 12 ± 1.7637 (p <0.001) and 12.2 ± 2.4 (p <

0.0001) days of age, equivalent to 2.28 ± 0.71; 0.71 ± 0.28; 0.92 ± 0.34; 1.71 ± 0.25 and 1.74

± 0.34 weeks of age, respectively (Table 3.2). Taken together, the changes in heart rate,

SDNN, glucose, pO2 and urine flow rate occurred around the window of weaning time,

suggesting a relationship between weaning and these physiological changes.

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Variables Mean age (days) ± SE 1h rhythm lost 50.9 ± 4.8

Lowest actiwatch total activity 56.6 ± 4 Solid food was first consumed 29.6 ± 1.1

Table 3.1: The time window of weaning in lambs was defined as when lambs started to first consume solid food to when 1h rhythm due to suckling disappeared

Variables Breakpoint (days) ± SE (p) r2 Heart rate (bpm) 41.1 ± 5.5 (<0.0001) 0.6914

Arterial pressure (mmHg) 16 ± 5 (0.002) 0.2345 SDNN (msec) 51.6 ± 13.3 (0.0002) 0.473

SD Delta NN (msec) 60 ± 26.2 (0.0247) 0.3296 RMSSD 60 ± 26.2 (0.0247) 0.3296

LF power (nu) 3.6 ± 0.54 (<0.0001) 0.1578 HF power (nu) 2 ± α (1.0) 0.2588

LF/HF ratio 5 ± 2 (0.014) 0.1417 Glucose (mmol/l) 61.1 ± 7.9(<0.0001) 0.8184 Lactate (mmol/l) 6.5 ± 2.4 (0.0084) 0.2375

O2 sat (%) 99 ± 29.5 (0.0012) 0.3133 pO2 (mmHg) 52.3 ± 22.3 (0.0213) 0.2738

pCO2 (mmHg) 12 ± 6.4 (0.066) 0.1119 pH 55.5 ± 80.7 (0.494) 0.0223

HCO3 (mmol/l) 99 ± 68.4 (0.1522) 0.0426 BE (mmol/l) 73.6 ± 38.5 (0.0595) 0.1609

Hb (g/l) 12 ± 1.7637 (<0.001) 0.2814 Cortisol (ng/ml) 12.2 ± 2.4 (<0.0001) 0.414

Urine flow rate (ml/min/kg) 38.4 ± 8.7 (<0.0001) 0.5 Table 3.2: Break points of cardiovascular, metabolic, endocrine and renal variables. Break point is the point (day of age) where the slope of the plots of the variable versus age changes.

83

3.4 Discussion:

The main findings of this study are that there were sex differences in pH value and LF

power at 3 days of age and in heart rate at 6 months of age. Cardiovascular function in the

lambs changed over time with a decrease in heart rate and increase in arterial pressure

together with the maturation of the autonomic system (i.e., increase in the time domain of

HRV and a reduced influence of the sympathetic system on the heart as indicated by a

decrease of LF power and therefore LF/HF ratio). The relation between HRV frequency

domain variables and autonomic function will be discussed further below.

In addition, we also found the acid base balance shifted toward the alkaline range

over age with a decline in pCO2, and increase in pH. The efficiency of pulmonary oxygen

uptake indicated by arterial PO2 was progressively increased after birth until 3 months of ages

and slowly increased thereafter which suggested that a process of improvements in

pulmonary gas exchange still continued at least for the first 3 months of life. Glucose, lactate

concentration, urine flow rate and Hb decreased with increasing age, while a slight anemia

occurred at 1 month of age. The cortisol concentration was attenuated a few days before

birth, decreased rapidly for the first 10 days of age and gradually thereafter.

The 1h rhythm that exists in the lambs but not in the adult sheep is associated with

suckling. The disappearance of 1h rhythm was associated with the changes in growth rate

and the decline in total activity. The break points of some physiological variables (including

changes in heart rate, SDNN, glucose, pO2 and urine flow rate) occurred around the window

of weaning time suggests the effects of weaning and thus the beginning of solid food intake.

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Indexes for HRV analysis are usually obtained by linear methods (including time

domain and frequency domain analyses) or nonlinear methods. In this study we used the

linear method with statistical indexes of the time domain (SDNN, SD Delta NN and

RMSSD) and frequency domain indexes. Compared to the time domain, the frequency

domain provides better information of the sympathetic-vagal balance on the heart since the

SDNN of time domain represents both sympathetic and parasympathetic activity, but does

not allow for identification of changes in HRV elicited by autonomic system. Frequency

domain analysis, on the other hand, is composed of several oscillatory components reflecting

the autonomic system: high frequency (HF), low frequency (LF) and very low frequency

(VLF) components. HF covers a frequency from 0.15 to 0.4 Hz and is an indicator of the

influence of the vagus nerve on the heart. LF, ranging from 0.04 and 0.15 Hz, represents both

vagal and sympathetic components on the heart, with a predominance of the sympathetic

system. Therefore, the LF/HF ratio reflects the relative changes between sympathetic and

parasympathetic systems, and hence represents the sympathetic-vagal balance acting on the

heart. VLF does not have important role in indicating the function of the autonomic system

(Vanderlei et al., 2009).

The possibility that the surgery could affect my measurements of cardiovascular

function, in particular the postoperative autonomic nervous system hyperactivity (i.e.

increased LF power), was considered. However, I do not think that this is the case for several

reasons. First, the surgery for catheter implantation in our lambs was a simple procedure

performed under both general anesthesia and local analgesia. Second, the cortisol

concentration measured on day 3 (i.e day after surgery) tended to be lower than that prior to

surgery, suggesting that the surgery was not too stressful event compared to birth itself.

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Third, the values of heart rate, arterial pressure and HRV in our study were similar to those

reported in other studies which focused on the postnatal changes in cardiovascular function

in lambs. Consistent with our results, other studies also found a similar high heart rate after

birth, a decrease of heart rate and increase of arterial pressure at rest in lambs from 1 day to 6

months of age (Klopfenstein & Rudolph, 1978; Minoura & Gilbert, 1987). The increasing

trend of beat to beat HRV in my study from postnatal day 2-3 to 1 year of age was similar to

other findings in lambs from 1 to 10 week-old even though the HRV in lambs in the study of

Siimes et al seemed to plateau earlier (after 6 weeks of age) (Siimes et al., 1984). In the

human, it has also been shown that HRV increases with gestational age, postnatal age and

with maturity (Harper et al., 1976; Katona et al., 1980; Wheeler et al., 1979).

The decline in heart rate values with ages observed from our study may result from an

increase of parasympathetic activity or a decrease in sympathetic activity or from

simultaneous changes in both regulatory systems. The latter is most likely the case, since the

two branches of the autonomic nervous systems do not function on a continuum to regulate

cardiac activity, thus the effect of each system on the heart cannot be simply determined by

addition or subtraction of the relative components (Hainsworth, 1995). Instead, heart rate is

affected by the net interaction between vagal and sympathetic regulation. This can explain

the unsynchronized changes among LF power, HF power and heart rate over ages in our

lambs. The sympathetic system appears to be a major regulator of vascular function up to 8

weeks of life and by 3 months of age, vagal regulation becomes dominate (Gronlund et al.,

1989; Woods et al., 1977). Our results represent the normal postnatal ANS maturation in the

term lambs with a progressive decrease in heart rate and increase in HRV which is, in part,

consistent with findings in human newborns. (Longin et al., 2005)

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Changes in the time domain of HRV was inversely related to changes in heart rate.

(Fig. 3.4) When heart rate decreased, the time intervals between the single beats had to

become longer, thus, as expected, time domain of HRV increased. However, LF power which

mainly indicates the influence of the sympathetic system on the heart altered independently

to the changes of heart rate. The increase to peak of LF power at ~ 10 days of age in the

lambs was sex specific, due to significantly increased LF power in female from 3 days to 10

days of age but not in male. Also, this peak LF power at ~ 10 days of age occurred around

the same time with the peak of total activity score (at ~ 15 days of age) observed from the

actiwatch while heart rate slightly decreased at this age. It has been known that physical

activity strongly influences cardiac activity and is frequently characterized by decreasing

vagal tone and increasing sympathetic influences on the heart (Nakamura et al., 1993; Voss

et al., 2002; Warren et al., 1997). This was shown in the work of Voss et al in which heart

rate and HRV were evaluated in seven horses at rest and during six incremental levels of

exercise from walking on a dry treadmill to trotting in water above the elbow. Heart rate

increased together with a decrease in overall variability (SDNN, withdrawal of vagal), LF

power increased (higher influence of sympathetic nervous system) and HF power decreased

(decrease in parasympathetic activity) during exercise compared to rest. (Voss et al., 2002)

It was also found that LF power in male lambs was significantly higher than in female

lambs at 3 days of age, but there was no difference in heart rate. This highlighted the greater

sensitivity of HRV in detecting changes in sympathovagal balance in the absence of apparent

changes in heart rate or respiratory rate. Indeed, Sleigh and Henderson found HF power,

which represented vagal activity, reduced in relation to pre-surgery anxiety, whereas no

effect was seen on heart rate. (Sleigh & Henderson, 1995) The sex difference in LF power

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could be due to an influence of sex hormones on the ANS. Testosterone is present in the

plasma of male lambs by 36h after birth and increases to a peak at 3 days of age (Savoie et

al., 1981). The higher LF power in male lambs could also be a very first sign of sympathetic

nervous system response to an unfavorable environment. This could be related to the greater

degree of acidemia (lower pH) in the males than females at this age. In this case, the high

HRV response in males may reflect an appropriate adaptation. On the other hand, it could

also reflect a higher innate sympathetic drive in the male which leads to their higher risk of

metabolic syndrome, a situation observed in human adults. (Chang et al., 2010; Licht et al.,

2010) However, this is not likely to be the case, since insulin resistance, a key phenomenon

in the pathogenesis of many metabolic cardiovascular disorders, results from a diminished

regional blood flow and tissue glucose delivery as a consequence of vasoconstriction due to

an enhanced sympathetic drive (Esler et al., 2001) and we do not see any gender differences

in arterial pressure in lambs at this age and even later in life. Another possibility, even though

less likely, of high LF power in male lambs is due to the high body fat. In human, obesity is

associated with increased sympathetic outflow to the kidneys and skeletal muscle vasculature

(Esler et al., 2001) and body fat is the main source of sympathetic discharge. Even though

male lambs usually have a higher carcass weight than female lambs at the same age, this

difference is attributed to a heavier fat-free weight only (Bennett et al., 1991).

Interestingly, our results showed that male lambs had a lower pH value than females

at 3 days of age. Although the extent of acidosis in the males was not great, this could be one

of the predisposing factors that lead to a higher mortality rate in male than female neonates

(Khoury et al., 1985), especially related to respiratory distress syndrome (RDS) (Ingemarsson

et al., 1997; Ingemarsson, 2003; Khoury et al., 1985). Ishak et al (2012) have recently

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reported in the fetal lambs delivered by Cesaeren section at 133 days, arterial pH lower in the

male lambs than in female lambs in the initial postnatal period, with the sex difference (~0.15

pH units) being greater than the difference in the 3 day old lambs in my study (~0,08 pH

units). This suggests that an attenuated sex difference in arterial pH is present even in term

lambs. A study in Sweden analyzed acid- base balance in 15,480 human cord arterial blood

samples in term infants and showed a higher rate of pronounced acidaemia (pH < 7) in male

newborns (64%) than female newborns (36%) (Ingemarsson et al., 1997). In our study, the

earliest pH measurement obtained from the lambs was at 3 d, thus the male pH value might

have been even more acidotic at birth. This is especially a serious problem if we consider the

higher rate of preterm birth in humans, which is usually associated with pulmonary

immaturity in male infants compared to females (Ingemarsson, 2003). In addition, there is a

delay of lung maturation in male fetuses (Torday et al., 1981) and antenatal betamethasone

administration is not as effective in preventing RDS in male as in female. (Ballard et al.,

1980; Papageorgiou et al., 1981)

A sex difference in heart rate was also observed in lambs at 6 month of age. Heart

rate was significantly higher in male than female. Experiments of this age group in our study

were conducted on postnatal day 195.2 ± 8.6 and 214.6 ± 4.2 in females and males,

respectively. Female lambs raised under natural light can reach puberty as early as 153 ± 7.1

days in Dorset (Fitzgerald & Butler, 1982) or up to 234 days in Dorset and 245 days in

Suffolk ewe lambs (Quirke et al., 1985). Male lambs (Dorset x Leicester x Suffolk) raised

indoor under natural light conditions, on the other hand, enter puberty a bit earlier between

150 to 200 days of age as defined by an increase in plasma testosterone concentration from

1ng/ml at 150 days of age to 8 ng/ml at 200 days of age (Yarney & Sanford, 1989). Since our

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lambs were raised under a 12 hour light/dark cycle, their sexual maturation could be delayed,

but in any case male lambs reach puberty earlier than females. Therefore, the differences in

their heart rate might be a result of sex steroid hormone influences on the cardiovascular

system. With the growing rates of cardiovascular disease and metabolic syndrome around the

world, they could become the epidemics of the century (Banos et al., 2011; Iyer et al., 2011,

Barouki et al., 2012). There will no doubt be increasing attention paid to the effects of sex

hormones on the occurrence of these diseases. It appears that androgen and estrogen exhibit

differential effects on the pathophysiology of the vascular beds in male and female. So far,

whether testosterone is protective or harmful to the development of those chronic diseases is

still a matter of debate (Corona et al., 2011; Traish & Kypreos, 2011). In my study, it is

uncertain if a gender difference in heart rate would have any long-term effects on the lambs

given that there were no accompanying significant differences in other systemic

cardiovascular variables such as blood pressure and heart rate variability between genders at

this age.

The rapid increase in arterial PO2 in the postnatal lambs compared to the fetus at term

is the result of a transition from gas exchange via the placenta in the fetus to air-breathing in

the newborn. As the lung takes over the role of placenta in gas exchange, it is necessary that

the lung fluid is cleared from the airway and pulmonary vascular resistance decreases around

birth (Hooper & Harding, 2001). However, my results showed that after birth, arterial PO2

continues to increase slowly with age. This increase in PO2 is consistent with a slow increase

in O2 saturation and slow decrease in PCO2. These changes could reflect slow changes in

alveolar O2 and CO2 concentrations in the lung. If this is the case, the lambs must be

hypoventilated initially but increase thereafter due to a slow shift in the set point for

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ventilatory control in the carotid body regulating PO2. Alternatively, these changes could

reflect slow changes in the alveolar-arterial gas differences. In that case, the lambs are

ventilated adequately at first but there is a diffusion limitation that is slowly corrected over

time. This is most likely to be the case since it has been shown that in sheep even though the

blood air distance becomes quite thin after birth (~0.2µm), it is only at focal points initially

and it is not until they reach adulthood that the basement membrane fusion has spread

enough to cover more than 50% of the epithelial surface (Hooper & Harding, 2001).

The level of activity in the lambs increased progressively right after birth, reached a

peak level at ~13-15 postnatal days and then started to decline, becoming stable around

postnatal day 30. The activity results for the first 10 days in these newborn lambs is similar to

the activity level that was found in term lambs in a previous study (Rurak et al., 2008). Over

the 90 days of monitoring, even when lamb’s activity decreased to a basal level, the lowest

average values of their total activity score (212473 ± 23298) which is on postnatal day 60

was still higher than the value reported in adolescent sheep (100927 ± 7189) or in adult ewes

(134808 ± 14644) held in the same conditions (Rurak et al., 2008). To determine when the

level of activity decreases to the level of adolescent or adult sheep will require a longer

monitoring period.

For the first 30 days, the increase in daily total activity score, active bouts per day and

the intensity of activity to the peak on postnatal day 13-15 of is probably a sign of the

maturation in their bone and skeletal muscle after birth (Rurak et al, 2008). The pattern is

reversed for the duration of activity. The shifts in activity pattern may reflect the suckling

pattern in lambs which goes from less frequent and intensive for a longer period of time at

birth to more frequent and intensive but for a shorter periods of time around postnatal day 15,

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and then suckling less and less until eventually the lamb was weaned and eating solid food,

which they can do for longer periods of time. The underlying mechanism of this pattern of

activity may involve the changes in the hormonal levels, such as insulin-like growth factor I

which peaks at around the same time (i.e postnatal day 13-15) (Greenwood et al., 2002),

since from direct observation, lambs start to be interested in grain at around this period.

Indeed, a study that was focused on the effects of birth weight and postnatal nutrition on

neonatal lambs has shown that the IGF-I concentration is maximum on days 9-11 in high

birth weight lambs and on days 11 to 13 in low birth weight lambs (Greenwood et al., 2002).

Another explanation for the shifts in lambs’ activity could be due to the changes in milk

production from the ewe over time. When the milk production from the ewes is at its peak,

the lamb’s activity was also highest. Milk production of 2 year old ewes rearing twin lambs

and 1-2 year old ewes rearing singletons peaked at 21 day of lactation and 27 to 30 of

lactation, respectively (Cardellino & Benson, 2002). However, the time of the peak of milk

production in our ewes could have been shorter, at around 15 day of lactation, since most of

them werer older than 2 years.

Using FFT we found the initial rhythms present after birth in lambs was dominated by

the short 1-2 hour rhythms, which corresponded to suckling patterns from our DVR

observations. Over the next four weeks, these rhythms decreased in dominance, matching the

decrease in frequency and duration of suckling. When lambs’ nutritional requirement

changed from the solely milk regimen to solid food supplement, reflected by the

disappearance of the short 1-2 hour rhythm due to suckling, their activity also declined.

Interestingly, around the time when the 1-2 hour rhythms disappeared and their activity

declined, their growth rate was also reduced.

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In human infants, there are ultradian rhythms of 3h, 4h and 6h as sleep, wake and

food intake behavioral cycles (Menna-Barreto et al., 1993). The time course of the

development of a permanent circadian rhythm for lambs seems to be similar to that found in

humans. In humans, Tomioka & Tomioka found that the circadian rhythm of sleep-wake

cycle appeared in the first week of life in 3 infants and became dominant in the 3rd and 5th

week of life (Tomioka & Tomioka, 1991). However, there was a large interindividual

variability of both the ultradian rhythms and the development of a 24 h pattern, whose

appearance varied considerably between the 6th and the 16th week of life (Lohr &

Siegmund, 1999). In sheep, the dominant circadian rhythm is in synchrony with the

development of a postnatal melatonin rhythm in lambs at 3-4 weeks (Nowak et al., 1990). In

some lambs in our study, the circadian rhythm existed for 1-2 days after birth and

disappeared for almost a month before it became persistently dominant. The existence of the

circadian rhythm for the first few days after birth in the lambs could be an influence of the

remaining melatonin level from the ewe after birth (McMillen et al., 1989).

Similar to infants, lambs have 2 developmental processes in the development of their

circadian rhythm: a decreased ultradian and increased circadian components and the process

of synchronization with external time cues or social factors such as light/dark cycle, meal

feeding, experiments, ect. The development of circadian rhythm appears to be important,

since its existence plays an important role in the well being of both animals and humans,

because it guarantees optimal functioning of biological systems with maximum efficiency.

Once the circadian rhythm appears, it lasts for a considerable length of time, but begins to

break down during illness or old age (Weinert, 2000).

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The eight hour ultradian rhythm emerged as a dominant rhythm around 2 weeks after

birth and is thought to be due to the daily scheduled feeding times, which occured twice a

day at 7:30-8:00 am and ~ 2:30-3:00 pm, an approximately eight hour interval. Even though

the young lambs only fed on their mothers’ milk, they became more active as their mothers

were being fed. To further confirm that the 8 hour ultradian rhythm is the result of the

feeding time entrainment, further study is needed to determine if this ultradian rhythm exists

in the lambs that develop under field conditions where they can graze ad libitum.

The 6h rhythm seemed to become the third most dominant rhythm when the lamb was

about two months of age. This 6 hour rhythm was most likely a result of maturation of the

lambs’ rumen and reticulum, which were stimulated by the intake of solid food, as we found

a correlation between the first time lambs eat significant amounts of hay, and the day this 6 h

rhythm became dominant. This is supported by research which showed that β-

hydroxybutyrate production from butyrate in isolated sheep ruminal cells increased 10 fold

from day 42 to day 56, as compared to the rate at birth; this marks a shift in the ability to

synthesize ketones in a mature rumen, and the development of the rumen microbiota

(Baldwin & Jesse, 1992). Lane et al also observed changes in the morphology of the rumen

epithelium (fewer and larger intraluminal papillea per cm2) on day 84 from lambs fed with

solid food after postnatal day 49 (weaned lambs), as compared to epithelium of lambs fed

solely with milk replacer (non-weaned lambs) (Lane et al., 2000)

The suckling frequency was similar in singleton, twins or triplet lambs. However,

triplets seemed to have longer feeding bouts which might be secondary to the restriction of

the available milk supply in triplet pregnancies. As a result, twins or triplets spent more time

in competing with their siblings to obtain milk, which led to an increase in their feeding

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bouts,but it did not appear to improve the efficiency of their suckling, as reflected in the

tendency for a lower weight gain in triplets and twins, as compared to the singleton, even

though this difference was not significant. This could be due to my small sample of triplets.

Results arising from a much larger study, comprising approximately 3000 singleton, 1500

twins and 200 multiple lambs have shown that singleton lambs gained significantly more

weight than twins and multiples. As a result, singleton lambs were heavier than those born as

twins and multiples, both at birth and at weaning (Carrillo & Segura, 1993).

The short 1h rhythm was not observed in adult sheep and only the lactating ewes have

the short ~ 1h rhythm, but none of them had this as the first dominant rhythm. This suggested

that the 1h rhythm is the rhythm that the ewes share with their offspring. It has been known

that suckling serves as a mechanical stimulus to inhibit the HPA axis in the ewe to regulate

the estrus cycle. Depending on breed, time of the year and degree of mammary stimulation,

the duration of lactational anestrus varies considerably (Fletcher, 1971; Gordon & Siegmann,

1991; Mallampati et al., 1971). Usually after the termination of lactation, the mother and

lamb relationship does not fade away completely. Indeed, lambs remain associated with their

mothers for several weeks or months (Hinch et al., 1990). Therefore, beside measurement of

maternal prolactin level, the waning of this 1h rhythm in the ewe, which can be non-

invasively obtained by actigraphy, could be an alternative indicator that the ewe was free

from the lactational inhibition of reproduction.

Our results provided a normative data set of cortisol concentration in the normal

lambs at different ages under basal conditions. Cortisol level on postnatal day 2 ± 0.2 (i.e

prior surgery) were higher than that measured in the fetus at 7.1 ± 1.6 days before delivery

(at gestational age = 135.4 ± 0.7 days). This was consistent with the cortisol surge resulting

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from activation of the fetal HPA axis at ~ 125-130 of gestation which is required for the

initiation of parturition (Wood, 1994, Norman et al., 1985). Cortisol concentration declined

significantly thereafter during the first 10 days of life and was returned to the similar level

observed in the fetus on gestational day 135.4 ± 0.7, and further decreased after 1 month of

age to reach the adult value at 3 months of age. However, plasma ACTH concentration was

not well-correlated with the postnatal plasma cortisol changes. ACTH was low on postnatal

day 2 ± 0.2, probably due to a refunctioning of cortisol negative feedback after birth, which

was desensitized at the end of gestation. In addition, the high cortisol concentration without

an adequate increase of ACTH could be a result of increasing cortisol binding protein near

term, which exaggerates the total cortisol level but suppresses the efficient negative feedback

of cortisol on the pituitary, as well as hypothalamus (Challis et al., 1995). The ACTH level

remained lower at other ages as well. It was not clear why there was dissociation between

plasma cortisol and ACTH concentration even at these later ages.

Cortisol concentration was significantly higher in the morning (at 10:00, 10:30 and

11:30 am) than in the afternoon (at 3:30 pm) or in the evening (at 9:30 pm) in the lambs at 10

days and 1 month of age which suggested that there was diurnal rhythm of cortisol in the

lamb at these ages. Such a rhythm was not found at other ages (3 day, 3, 6 and 12 month-old)

which is in agreement with the findings from the work of Bell et al, who reported there were

no endogenous circadian or diurnal rhythms in ACTH or cortisol secretion in pregnant, non

pregnant cycling, non cycling or ovariectomized adult ewes (Bell et al. 1991). In our study,

the samples were collected for only a 12h period; therefore we could not make a firm

conclusion about a circadian rhythm of these hormones, which appear to be present in lambs

after 3 weeks of age (Parraguez et al., 1989). There were some other factors that might have

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contributed to the higher level of cortisol in the morning. First, it has been known that the

cortisol level increases after feeding in pregnant ewes (Slater & Mellor, 1981) and in the

human (Follenius et al., 1982). Lambs around 10 days to 1 month old had a high activity of

suckling. In addition, from direct observations we noticed that after the first week lambs

started to participate in nozing the ewe’s daily feed. However, if the higher cortisol level was

a response to feeding, the cortisol level measured at the earlier time (around 8:30 am) should

have been higher, and also higher at the pm feeding time (at 3:30 pm), which was not the

case. Thus, it is more likely that the high cortisol level for the 2 h of experiment after dosing

is a result of the lamb’s response to the social cue of the investigators’ close proximity for the

2 h duration when there was frequent sample collection. This social cue disappeared at later

ages when lambs become habituated to the investigators.

Although there was no rhythm in the older lambs, we did find a sex difference in the

cortisol level in lambs at 1 year of age at 0.5 h (10:00 am), 1 h (10:30 am), 6 h (15:30 pm)

and 12 h (21:30 pm) sample. Studies that have examined the influence of sex hormones on

the cortisol level have demonstrated estrogen-enhanced HPA axis function in adult animals

under both basal and stress conditions (Bell et al., 1991; Carey et al., 1995; Wood et al.,

2001). However, my results showed that basal cortisol concentration was lower in the female

than male lambs, which suggests a reduced HPA axis activity in the female at this age.

Nevertheless, since cortisol levels vary during the estrous in sheep (Bell et al., 1991), it is

possible that the cortisol levels in the female were measured in noncycling animals or during

anestrus cycles which have no estrogen effects.

In our study the lambs were not weaned abruptly. In studies focused on the effect of

diet and weaning on growth, the time of weaning can be as early as 28 days of age (Lane &

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Albrecht, 1991) or as late as 42 days, by which time the rumen is believed to be already

functional (Holcombe et al., 1992). The earliest our lambs were separated with their mothers

was at 47 days of age and in some cases they remained with their mothers for up to 80 days.

However, the lambs were gradually weaned by the ewes refusing to let them suckle and

walking away from them, even though the lambs still tried to nurse sometimes. In the context

of our study, the time of weaning was defined as the time window when lambs started to first

consume solid food (hay) to when the 1h rhythm due to suckling disappeared.

The blood glucose concentration, which is related to rumen development (Holcombe

et al., 1992) decreased gradually with age, and the value at 1 month was significantly

different from that at other ages. This alteration in glucose concentration were consistent with

our observations of the time lambs first started to consume solid food (29.6 ± 1.1 days of

age) which indicated a functional forestomach already developed. Similar results were also

reported in the work of Holcombe et al., (1992). Our results, taken with the data from the

Holcombe et al study, suggest that the window of weaning is around 1 month of age. It

appears that the greatest changes (i.e break points on piecewise linear regression) of most

physiological variables (heart rate, SDNN, Glucose, pO2, urine flow rate) occurred around

the window of weaning time (1 to 2 months of age), so that by 3 months of age they are

similar to their levels at more mature ages.

A decrease in urine flow rate around the time of weaning was reasonable as the lambs

changed from a predominantly milk diet to a solid food diet. In addition, it has been known

that feeding activity has effects on the cardiovascular system; thus it was not surprising to

observe heart rate and HRV (SDNN) were significantly altered at the time of weaning.

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Overall, our results revealed a data set of normal development of physiological,

endocrine and behavior characteristics in the lambs in relation to several milestones of the

developmental process (birth, weaning and puberty) under basal conditions. This will provide

reference information of these variables for future studies related to any relevant disorders.

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4. Acute Fluoxetine Effects In The Newborn

4.1 Introduction:

Major depression is probably one of the most burdensome disorders in the world. In

the report of World Health Organization (WHO) in 1996, it was estimated that by 2020

depression will rank second only after cardiovascular disease in terms of its disability-

associated burden (Murray & Lopez, 1996). Depression affects at least about 15% of women

in their lifetime (Burt & Stein, 2002; Kessler et al., 2003). The risk is even higher during the

reproductive years. Thus there is a significant risk that one might become depressed during

pregnancy or even get pregnant while being depressed or on antidepressant therapy. Recent

studies have failed to confirm the previous belief that the state of pregnancy is protective

against mood disorders (O'Hara et al., 1990). Moreover, it has been shown that untreated

maternal depression leads to long-term neurobehavioral consequences in the offspring,

including poor cognitive function, and diminished language and psychomotor development

(Deave et al., 2008; DiPietro et al., 2006; Hay et al., 2008; Murray & Cooper, 1997; Murray

et al., 2001; Nulman et al., 2002). Therefore, pharmacologic treatment is recommended for

pregnant women who have moderate to severe symptoms.

In addition, depression during pregnancy is in turn a risk factor for postpartum

depression, affecting 10% to 22% of women (Burt & Stein, 2002). Since the postpartum

period is the most vulnerable time for serious symptoms and their short-term and long-term

consequences to not only the mother but also the entire family unit, this has resulted in

postpartum depression becoming a significant health care problem. Moreover, it has been

shown that the risk of relapse increases 5 times in women who discontinued medication

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during their pregnancy compared with those who maintained their medication (Cohen et al.,

2006). Thus continuing medication in the third trimester is recommended (Toohey, 2012).

Among the classes of antidepressants, SSRIs have been widely used in pregnant women due

to their relatively safe side effects profile. In fact, 33% of pregnant women with clinical

depression were prescribed an antidepressant (Oberlander et al., 2006), in which SSRIs

accounted for 75% of the cases (Simon et al., 2002). Recent studies have reported an

increasing use (4.9- 6.2% increase over 9-14 years) of SSRIs during pregnancy (Andrade et

al., 2008; Cooper et al., 2007; Simon et al., 2002; Wichman et al., 2008). However, third

trimester use of SSRIs, unfortunately, was also reported to increase the risk of a variety of

neonatal complications termed “neonatal behavioral syndrome” or “poor neonatal

adaptation” after birth (Chambers et al., 1996; Cohen et al., 2000; Kwon & Lefkowitz, 2008;

Mhanna et al., 1997; Oberlander et al., 2004; Oberlander et al., 2006), a phenomenon

observed in 10% to 30% of infants exposed in utero to SSRIs (Koren et al, 2009). The

presenting symptoms include irritability, persistent crying, shivering, tremor, restlessness,

feeding difficulties, sleep disturbances or even more severe symptoms, such as seizures,

dehydration, excessive weight loss, hyperpyrexia and the need for intubation (Moses-Kolko

et al., 2005). Even though this syndrome is self-limiting, it results in longer hospitalization

and led to a warning from Health Canada and FDA about the adverse events when using

SSRI/SNRIs in pregnant women during the third trimester (Summary minutes of the

pediatrics subcommittee of the anti-infective drugs advisory committee.2004).

There are several suggested mechanisms for the poor neonatal adaptation. First is a

withdrawal syndrome due to the discontinuation of maternal-fetus placental transfer right

after birth. Second is a serotonin toxicity mechanism induced by the high circulating SSRI

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concentrations in the newborn at birth, due to their low capability of drug clearance. Third is

an SSRIs-elicited alteration in fetal brain development. Findings supporting the latter

mechanism arise from the work of our group (Morrison et al, 2001; Morrison et al., 2002;

Morrison et al., 2004; Morrison et al., 2005), which reported a decrease of fetal rapid-eye-

movement (REM) sleep in lambs whose mother were administered FX i.v. continuously for 8

days in late gestation. Similar findings were also demonstrated in human fetus with SSRI

exposure (Mulder et al, 2011). Further supporting evidence comes from the study of

Oberlander et al., 2006 and Rurak et al., 2011, who observed altered fetal middle cerebral

artery flow characteristics in 36 week human fetuses exposed in utero to SSRIs compared to

a control group.

However, which mechanism is responsible for the observed poor neonatal adaptation

is not yet clear. Currently, the management for these syndromes is simply symptomatic with

supportive care (Koren et al., 2009). Since the strategy of treatment could be completely

opposite for the first and second mechanisms mentioned above, it is important to elucidate

the underlying mechanism for the syndrome. For this reason, in this study we investigated the

effect of acute FX, one of the most prescribed SSRIs on growth, behavior, and

cardiovascular-respiratory, and endocrine functions in ~4 d old newborn lambs. The

hypothesis that was tested was that acute administration of FX to achieve plasma

concentrations similar to those in human newborns exposed to FX in utero would result in

effects similar to poor neonatal adaptation in human infants.

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4.2 Methods:

4.2.1 Experimental Protocol:

21 lambs were included in the study reported in this chapter, of which 9 lambs were

in the control group with sterile water i.v. bolus administration (0.1ml/kg) and 12 lambs were

in the FX group with FX (1mg/kg) bolus i.v injection, respectively. The animal housing,

surgical, experimental and analytical methods are described in Chapter 2. Heart rate and

blood pressure data used in this chapter were averaged at 1 min intervals for 15 mins before

dosing and the first 10 mins after dosing and at 5 min intervals for the remaining time while

HRV data were averaged over 30 mins intervals and also averaged at 1 min intervals for 2

min before dosing and 5 mins after dosing. Blood gas samples were collected at -15, 5, 15,

30, 60, 120 mins as described above, however, another arterial blood gas sample was taken at

2 mins in 4 lambs in this study to characterize more fully the short term changes in blood gas

and acid-base status following water or FX administration. For the rest-activity analysis,

beside the control group with sterile water injection and FX group with FX injection, another

group of normal term lambs in which no surgeries or experiments occurred were included.

4.2.2 Statistical Analysis:

Activity data were analyzed using a 2 way GLM for repeated measures ANOVA

followed by the Bonferroni t-test for multiple comparisons versus the control group to

determine the effect of treatment and sex on changes in behavioral activities. Cardiovascular

and blood gas data were analyzed using a 3 way ANOVA followed by Bonferroni’s

correction for pairwise or control comparisons to determine the effects of treatment, time and

sex on the measured variables. Two way GLM for repeated measure ANOVA was used for

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not-normally distributed variables (SDNN, SD Delta NN, RMSSD, LF power, HF power,

LF/HF ratio, O2 saturation, pCO2, glucose and Hb) to determine the effects of treatment and

time. Because plasma cortisol concentrations were not normally distributed, the non-

parametric Mann-Whitney U-test was used for the cortisol data to determine the effect of

treatment. P < 0.05 defines statistical significance. Data are presented as mean ± SEM.

4.3 Results:

On postnatal day 3.3 ± 0.4 and 4.2 ± 0.4 sterile water (0.1ml/kg, N=9) or FX

(1mg/kg, N=12) was acutely injected via the jugular vein catheter, respectively.

4.3.1 Plasma FX Concentration And Weight Gain:

The plasma FX concentration in the ~ 3 day old lambs over 72h of experiment and in

13 individual human infants sampled at postnatal day 2 following in utero exposure to the

drug cover a similar range (~ 10-100 ng/ml) (Fig. 4.1A). (Chow, 2013; Kim, 2000) There

were no significant differences in postnatal weight gain on postnatal day 4 between the

normal term lambs group who were not subjected to any experiments or surgery (0.327 ±

0.048 kg, n = 11), control group (0.411 ± 0.056 kg, n = 9), FX group (0.392 ± 0.045 kg, n =

12), respectively (Fig. 4.1B). This indicated FX does not have any effects on initial postnatal

weight gain in the neonatal lambs.

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FIG. 4.1: (A) Plasma FX concentration verus time curve in the 3 day-old lambs (closed symbols and solid line) and in 13 individual human infants (open symbols) sampled at postnatal day 2 following in utero exposure to the drug. Human data was taken from the dissertation of Dr. John Kim, 2000 and the lambs data was taken from the dissertation of Dr. Tim Chow, 2013. (B) Weight gain on ~ postnatal day 4 among the normal term lambs (n = 11, open bar), control group (n = 9, black bar), FX group (n = 12, gray bar) and post FX day 1 (n = 12, light gray bar)

Time (h)

0 20 40 60 80

[FX

] (ng

/ml)

1

10

100

1000

3 d lambsHuman PKU [FX]

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4.3.2 Rest Activity Cycles:

The number of active bouts per day, total activity score, the intensity and duration of

activity and the percent of time moving and percent immobile time were comparable between

the normal term lamb group (n = 10), the control group (n = 6) and the FX group (n = 11)

(Fig. 4.2). In addition, from the DVR observation we did not notice any obvious behavioral

changes or any symptoms representing serotonin toxicity with FX dosing. During the first

2.5h of experiment when lambs were kept in the sling, they just lay quiet or slept for most of

the time, and there were no obvious behavioral changes following FX administration.

4.3.3 Cardiovascular Variables (Heart Rate, Arterial Pressure, Heart Rate

Variability):

Heart rate, arterial pressure and heart rate variability (HRV) in the newborn lambs

with placebo and FX injection are illustrated in Figures 4.3 and 4.4, respectively. The time

points included in the 3 way ANOVA test (treatment x time x sex) were at -15 min, 1,2,3,4,5

and 10 min only, since the data was not normal distributed with additional time points.

Arterial pressure were significantly different between the control and FX group at 1, 2, 3, 4, 5

and 10 minutes post-FX (p<0.001), but not at the control time point (-15 minutes). The

maximum increase in arterial pressure in the FX group was 8.5 ± 1.1 mmHg at 4 minutes

following FX injection compared to values before FX injection. Heart rate was lower in the

FX group than the control group only at 2 minutes post-FX (p = 0.034), the difference was 38

± 9 bpm. However, these effects were very transient and self-limited since heart rate returned

to normal values within 1 min later and by 15 minutes post-FX arterial pressure had also

returned to values similar to those observed before FX injection.

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FIG. 4.2: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in the normal ~ 4 day-old term lambs (n = 10), control = on day of control experiment (n=6), and FX lambs on the day of FX experiment (n = 11) followed up to post FX day 1 (n = 11), post FX day 2 (n = 11) and post FX day 3 (n = 11). No significant changes were found between activities after FX administration and sterile water injection or normal day without experiments.

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FIG. 4.3: Arterial pressure and Heart rate (at 1 min intervals for the first 5 min after dosing and at 5 minute interval for the whole 2.5h of experiment) in the ~ 4 days old lamb with sterile water injection (control group) and FX injection (FX group). a indicates significant differences between variables at -15 min vs 1,2,3,4,5,10 min after FX within the FX group b indicates significant differences between control and FX at the corresponding time

a a a a ab b b b b b

b

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FIG. 4.4: Time domain of HRV (SDNN, SD Delta NN, RMSSD) and frequency domain (LF power, HF power, LF/HF ratio) at 30 min intervals in the newborn lambs at ~ 4 days old in the control group (open diamond and dotted line) and FX group (close circle and solid line) a indicates significant differences between variables at -2 min vs 1,2,3,4,5 min after FX within the FX group b indicates significant differences between control and FX at the corresponding time

a b b

a b b

a b b

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The time domain of HRV including SDNN, SD Delta NN, RMSSD, which is related

to the RR interval variability was significantly higher in the FX group than that in the control

group at 1 and 2 min after FX administration but were comparable between the two groups at

other time points. Indeed, there were significant relationships between the changes in SDNN,

SD Delta NN, RMSSD at 1 min following FX injection and the changes in heart rate at 2

mins following FX with r2 = 0.8, p = 0.001 and r2 = 0.57, p = 0.0017, respectively (Fig. 4.5)

However, the frequency domain of HRV including LF power, HF power and LF/HF ratio,

which indicate parasympathetic and sympathetic influences on the heart were not different

between the control and FX group and also did not change significantly over time. (Fig. 4.4)

Sex differences were observed with SD Delta NN, RMSSD (within the control group, at - 30

min) and with LF power and LF/HF ratio (at - 30, 60 and 120 min) but the differences were

not related to treatment (data not shown).

4.3.4 Arterial Blood Gases:

Figure 4.6 and 4.7 illustrate the changes in arterial blood gas variables (O2 sat, pO2,

pCO2, pH, BE, HCO3), and lactate, glucose and Hb concentrations in newborn lambs with

placebo and FX injection. There were no consistent changes in these arterial blood gas

variables in either control or FX group with time. Arterial pH tended to decline in both

groups with treatment but this was not statistically significant. A paired-t test was performed

to further compare Po2 and O2 saturation at -15 minute before and 2 minute after FX injection

in a smaller group of lambs (n= 4) but no statistical significance was reached. There was a

sex difference in pH, HCO3 and lactate but this did not depend on the treatment. Hb was

slightly lower, but significantly on the day of FX than the control day. This could due to the

blood volume lost for sampling.

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FIG 4.5: The relationship between changes in heart rate at 2 mins following FX (delta HR) and changes in time domain of HRV (SDNN, SD Delta NN, RMSSD) (i.e Delta SDNN, Delta SD Delta NN, Delta RMSSD) at 1 min following FX in the lambs at ~ 4 days of age.

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FIG. 4.6: Arterial blood gases (pH, O2 saturation, pO2, pCO2, HCO3, Base Excess) in the newborn lambs (~ 4 days old) in the control group (open diamond and dotted line, n = 9) and FX group (close circle and solid line, n = 11)

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FIG. 4.7: Glucose, Lactate and Hb concentration in the newborn lambs (~ 4 days old) in the control group (open diamond and dotted line, n = 9) and FX group (close circle and solid line, n = 11)

4.3.5 Plasma Cortisol Concentration:

No differences were found between the cortisol concentrations in the control group

compared with the FX group as shown in Figure 4.8 (p = 0.925)

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FIG. 4.8: Plasma cortisol concentration in the ~ 4 days old lambs in the control group with sterile water injection (n = 8, open bar) and the FX group with FX injection (n = 5, close bar) at 30 and 15 minutes before injection and 0.5, 1, 2, 6, 12 hours after injection

4.4 Discussion:

We have found that acute FX administration did not cause any significant effects on

the feeding behavior, rest-activity cycle, cardiovascular-respiratory functions and the cortisol

level in the approximately 4 day old postnatal lambs. Mild and transient increase in arterial

blood pressure and decrease in heart rate occurred immediately after FX administration and

recovered within 15 minutes without any significant changes in HRV or arterial blood gases.

The plasma FX concentration time curve in our studied lambs over 72h of experiment

fell in the range of 10-100ng/ml, which is similar to the plasma FX level found in 13

individual human infants sampled at postnatal day 2 following in utero exposure to the drug

(Kim et al,. 2006). The wide range of plasma FX concentration in human infants could be

due to the differences in maternal FX doses or to cytochrome P450 polymorphisms in the

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infants, which could affect their ability to metabolize the drug, a phenomenon that has been

reported in adults (Bradford, 2002; Fjordside et al., 1999; Sachse et al, 1997; Scordo et al,

2005)

Acute FX effects on weight gain (feeding behavior):

Studies in humans regarding postnatal weight gain in infants breastfed by mothers

taking FX are inconsistent. While Chambers et al., (1999) reported a diminished postnatal

weight gain in nursing infants of mother treated with FX, a subsequent study from Hendrick

et al, (2003) failed to confirm this finding. In our study, the postnatal weight gain was

comparable between the acute FX exposed and the normal term lamb group on postnatal day

4. The activity bouts per day were also not different between the two groups. As was

demonstrated in chapter 3, this variable at this postnatal age was always associated with

suckling or suckling attempts. Collectively, this suggests that acute FX administration did not

affect feeding behavior in the lambs. However, it must be kept in mind that in our study, FX

was given acutely single dose, while in the human studies, the infants were exposed to FX

chronically from the prenatal to postnatal period. Acute and chronic FX exposure might exert

different effects on weight gain and feeding behavior. There are unchanged plasma

concentrations of leptin, a feeding-inhibiting hormone, in response to acute administration of

the serotonergic agent, clomipramine, in both normal weight and obese women (Oppert et al.,

1997). However, a study in rats showed acute or chronic FX administration resulted in a

significant decrease in food intake and plasma leptin levels in lean and obese rats (Dryden et

al.,1999). The differences in those findings and ours might be attributed to the differences in

animal ages or the dose of SSRIs used (10mg/kg in rat study and 1mg/kg in ours)

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In addition, there were also no significant differences in factors that might have

influences on postnatal weight gain in sheep such as birth weight, litter, breed, ewe-lamb

bonding and neonatal behaviors (Dwyer, 2003; Dwyer et al., 2005) between the normal term

lambs and the FX group. In addition, different from human studies, the effect of FX on

postnatal weight gain in sheep was not confounded by the severity of maternal depression

and other associated risk factors (eg: low socioeconomic status, family conflict, financial

stress, ect), which could presumably affect neonatal weight gain (Stewart, 2007; Wright et

al,2006; Zuckerman et al., 1989).

4.4.1 Acute FX Effects On Cardiovascular –Respiratory Functions:

FX and other SSRIs cause an acute increase in plasma serotonin levels through

inhibiting the serotonin reuptake by platelets (Maurer-Spurej et al., 2004). The effects of

serotonin on the cardiovascular system are contradictory between in vivo and in vitro studies,

at least in the rat (Watts & Davis, 2011). Serotonin has long been known as a vasoconstrictor

agent, which can lead to hypertension (Maurer-Spurej et al., 2004). However, it appears to

have divergent effects on the cardiovascular system. Some studies showed an increase in

blood pressure with acute FX injection (Anderson et al, 1996; Cavero et al., 1981;

Lazartigues et al., 2000) while others reported a decline in blood pressure (Callera et al,

2005; Fuller et al, 1979). In addition, the cardiovascular response to serotonin (i.v) is species-

specific (Watts & Davis, 2011): a decreased blood pressure was observed in broiler chickens

(Chapman & Wideman, 2002) while an increased blood pressure occurred in calves (Linden

et al., 1999). In conscious sheep, serotonin injection caused a dose-related increase in blood

pressure (Nelson et al., 1987). This is in agreement with our findings that arterial pressure

increased at 1-10 minutes after FX administration in the newborn lambs. Heart rate was also

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declined at 2 minutes post-FX. However, these effects were very transient and self-limited

since heart rate returned to normal values within 1 min later and by 15 minutes post-FX

arterial pressure was also returned to values similar to those observed before FX injection.

Therefore, we think these transient changes did not have any clinical significance in the

newborns. Interestingly, we noted that cardiovascular responses to acute and chronic FX

were opposite. The previous study from our group with 8 day i.v FX infusion in the pregnant

ewes showed an increase in fetal heart rate during the first 6h of FX infusion and no changes

in daily maternal arterial pressure and heart rate nor fetal arterial pressure during the period

of 8 day infusion (Morrison et al., 2002). The possible explanation for the different

cardiovascular response is that serotonin response is different with acute and chronic FX

treatment. It was reported in human and mice that acute FX treatment increases plasma

serotonin concentration while chronic FX treatment causes a decrease in plasma and platelet

serotonin levels (Alvarez et al., 1999; Alvarez et al, 1999). Indeed, in the previous sheep

study in our lab with chronic FX i.v infusion, the average maternal plasma serotonin level

increased for the first hour (at 15, 30 and 60 minutes) but decreased at 2 and 6 hours

compared to values prior to FX infusion (Morrison, 2001).

The transient mild bradycardia could be due to either a direct effect of FX on the

cardiomyocytes via 5-HT4 receptors or an indirect effect of FX on blood pressure leading to

a baroreceptor reflex in the autonomic nervous system (ANS) to increase vagal tone (Watts

& Davis, 2011). This reflex bradycardia could be mediated by 5-HT3 receptors located on

afferent vagal and sympathetic neurons (Kaumann & Levy, 2006). Even though it has been

reported that FX can inhibit the L-type Ca2+ current, which plays a role in reducing cardiac

contractility (Pacher & Kecskemeti, 2004), we believe that the bradycardia observed in our

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study is due to an ANS response on the heart rather than a negative inotropic effect of FX

since we did not notice any significant changes in the electrolyte concentration (i.e: K+, Ca2+,

Na+) at the corresponding time points (data in appendix 4).

Given the fact that bradycardia is a result of an ANS response, it is expected that

HRV changes accordingly. Indeed, the time domain of HRV including SDNN, SD Delta NN,

RMSSD was significantly higher in the FX group compared to the control group at 1 and 2

mins after FX administration. However, the effects appeared to be limited to only two

minutes after FX. As it was shown in Fig. 4.5, there was an inverse relationship between the

changes in heart rate and the changes of time domain of HRV. The greater the decrease in

heart rate at 2 min post-FX, the greater the increase in SDNN, SD Delta NN and RMSSD at 1

min post-FX. In addition, the frequency domain of HRV (LF power, HF power, LF/HF ratio)

was comparable between the two groups at all time points. Acute and chronic SSRIs

exposure might exhibit different mechanisms. A recent longitudinal observation human study

from our group has found a reduced fetal heart rate variability (decreased number of

accelerations) in the fetuses exposed to SSRIs throughout most or all of gestation, even after

controlling for maternal mood, which might reflect an alteration in prenatal cardiac

autonomic control (Rurak et al., 2011).

A FX-induced elevated arterial pressure and mild bradycardia at 2 minutes after FX

injection were associated with a very mild and transient hypoxemia reflected by lower

arterial Po2 and O2 saturation values observed at 2 minutes post-FX in a smaller group of

lambs (n = 4) , but these changes were not statistically significant. However, there were no

significant changes in arterial pH, Pco2 nor lactate concentration at 2 minutes following FX

injection. Moreover, Po2 and O2 saturation at 5 minutes post-FX and later time point were

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comparable with values observed before FX injection. Thus, transient hypoxemia with acute

FX injection was too minor to cause any respiratory acidemia. Collectively, respiratory

difficulties, a common feature of poor neonatal adaptation in human infants exposed to

SSRIs in utero, did not occur in the lambs with acute FX administration.

4.4.2 Acute FX Effects On HPA Axis Functions:

Similarly, FX did not cause significant changes in plasma cortisol concentrations in

the newborn lambs in our study. Also, no sex differences were found. To our knowledge,

there is limited information about the acute SSRIs effect on the neonate’s HPA axis as well

as the influence of maternal SSRI treatment on the fetal HPA axis. HPA axis function and the

central serotonergic system are known to be highly interrelated (Boadle-Biber et al., 1993;

Fuller, 1996a; Fuller, 1996b). In our previous sheep studies, 8-day late gestational FX i.v

infusion in the pregnant ewes resulted in an augmented prepartum rise in fetal ACTH and

cortisol levels and reduced low voltage rapid eye movement behavioral state in the fetus

(Morrison et al., 2004). In contrast, another human study from our group, which further

examined the long-term effects of prenatal SSRIs (fluoxetine) on the HPA axis in the

postnatal period, has shown an association between SSRI exposure and reduced basal cortisol

levels as well as an alteration in the HPA stress response pattern in the offspring at 3 months

of age (Oberlander et al., 2008). A lower cord blood cortisol level in SSRIs-exposed neonates

was also reported in a subsequent study (Davidson et al., 2009). Conflicting findings from

the sheep and human studies could be, in part, attributed to the differences in the timing and

duration of SSRI exposure. Even though there was no stressor stimulation included in our

study, it is unclear why the cortisol level in the lambs did not increase with acute FX

treatment as expected. It has been reported that acute administration of SSRIs increased the

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secretion of several hormones including cortisol and ACTH (Raap & Van de Kar, 1999)

while chronic treatment with SSRI (citalopram) resulted in a decrease (Jongsma et al, 2005).

Although the mechanisms are not fully understood, alterations in HPA axis activity in the

fetus might be one of the possibilities underlying negative perinatal outcomes observed in

human studies. In addition, this is particularly important in the context of fetal exposure to

excess glucocorticoids either by prenatal stress or maternal illness (e.g: depression, anxiety

disorders) or pharmacologic agents effects (eg: SSRIs, synthetic glucocorticoids for lung

maturation in preterm labor), which can program the HPA axis in ways that it will have

negative impacts on mental, metabolic and cardiovascular health later in life (Lupien et al.,

2009; Seckl, 1997; Tamashiro & Moran, 2010).

4.4.3 Underlying Mechanisms Of Poor Neonatal Adaptation:

There is an ongoing debate in the literature regarding the appropriate mechanism for

the observed poor neonatal adaptation, whether the symptoms reflect a true withdrawal

syndrome after abrupt discontinuation of transplacental transfer of maternal SSRI at birth or

are a result of serotonin toxicity due to SSRI exposure in the newborn (Haddad et al, 2005;

Isbister et al., 2001; Stiskal et al, 2001). Clearly, it is difficult to differentiate the syndrome

based simply on clinical signs and symptoms since the symptoms appear to be overlapped

between an SSRI withdrawal syndrome and serotonin toxicity. Thus, measurement of the

plasma concentrations of the administered SSRIs at delivery and at the time the neonate was

symptomatic would help in determining whether it is a withdrawal or toxicity. For example, a

high level of SSRIs concentration at the time of peak symptoms and a decline thereafter

would suggest a toxicity phenomenon. In contrast, if drug concentration is undetectable at the

time the symptoms are present, it is more likely a case of withdrawal syndrome. There is

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evidence supporting a withdrawal syndrome in the neonates after in utero SSRIs exposure

(Jaiswa et al., 2005). However, most of the withdrawal syndromes occurred more often with

paroxetine use than with other SSRIs (Jaiswal et al., 2003; Sanz et al., 2005), likely due to its

short half life and no active metabolite (Hiemke & Hartter, 2000). Since FX has a very long

elimination half life, it is is unlikely that there is a withdrawal syndrome with FX use. In

addition, there is no evidence for an ability to metabolize FX in the fetal lamb and human and

most importantly, it has been shown that the plasma FX concentration measured in human

infants at day 2 following birth is the same as that in cord blood at delivery (Laine et al.,

2003,Kim et al., 2006). Hence, it is less likely, we believe, that a withdrawal syndrome is an

underlying mechanism for poor neonatal adaptation after in utero FX exposure.

On the other hand, there is also evidence supporting the serotonin toxicity mechanism

in the “poor neonatal adaptation” in neonates who were prenatally exposed to SSRIs (Jessel

& Stiskal, 2004; Knopper et al.,2006; Kwon & Lefkowitz, 2008; Laine et al., 2003; Rampono

et al.,2004). These studies reported measureable concentrations of SSRIs (PX, FX and NFX)

in cord blood at birth and/or when the symptoms were present during the neonatal period. In

our study, we used a different approach to determine if serotonin toxicity is a mechanism

responsible for poor neonatal adaptation in the neonates. The study allowed us to reproduce

in the newborn lambs a similar drug level observed in the human neonates and the clinical

effects were recorded. As described above, the FX level in the lambs and in human infants

cover the same range. However, no symptoms related to poor neonatal adaptation (feeding

behavior, rest- activities cycle, irritability, respiratory difficulty) or other serotonergic

symptoms (restlessness, myoclonus, tremor, shivering) were observed in our exposed lambs.

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Thus, our results do not favor serotonin toxicity as an underlying mechanism for poor

neonatal adaptation.

To date, it appears that there is a growing body of evidence supporting the view that

SSRI-elicited alterations in fetal brain development may be responsible for poor neonatal

adaptation. As were mentioned above, supporting evidence include the initial study in sheep

from our group and subsequent findings in human which showed a decrease of fetal REM

sleep, an important determinant of brain development, in FX exposed fetuses (Morrison et

al., 2001; Mulder et al., 2011; Richardson, 1994). In addition, a recent study demonstrated

that the risk of respiratory distress, a common symptoms in poor neonatal adaptation, was

reduced by stopping SSRI treatment group before the end of pregnancy compared to the

group in which SSRI therapy continued until delivery, after controlling for maternal illness

severity, suggesting that the mechanism is less likely due to either withdrawal or toxicity

syndromes, (Warburton et al., 2010). Furthermore, it is known that serotonin plays an

important role in the regulation of early brain development. It autoregulates serotonergic

neuron growth in culture and the development of related neural systems (Whitaker-Azmitia,

2001). Thus, alteration the fetal serotonin level in the CNS secondary to prenatal SSRI

exposure could potentially influence serotonin signaling pathways in a developing brain,

affecting the fetal serotonergic tone and finally lead to changes in fetal/neonatal behavior

expressing as poor neonatal adaptation (Oberlander, 2012).

In conclusion, overall our results have shown that there is a lack of significant FX

effects on cardiovascular-respiratory, behavioral and endocrine functions in ~ 4–day old

postnatal lambs with acute FX administration. Thus SSRI toxicity is unlikely to be the

mechanism underlying poor neonatal adaptation in human infants exposed to this drug in

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utero. Further investigation is needed to determine the exact mechanism to provide more

sufficient information regarding the safety of FX and similar drugs in pregnancy

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5. Acute Fluoxetine Effects In The Postnatal Lambs (From 10 Days To 1

Year Of Age)

5.1 Introduction:

There is increasing awareness that children and adolescences also suffer from

depressive disorders, which were formerly thought only to occur in adults. In fact, the

prevalence of depression ranges from 0.4 to 2.5% in children and 0.4-8.3% in adolescents

(Birmaher et al., 1996; Fleming & Offord, 1990). And the cumulative incidence of major

depressive disorder (MDD) during adolescence is similar to the lifetime prevalence of MDD

in the adult population, which ranges from 15-20% (Birmaher et al., 1996). In addition,

teenage girls are at higher risk for developing depression than teenage boys, with a male:

female ratio of 1:2, similar to adults (Kessler et al., 1993; Cohen et al., 1993; Nolen-

Hoeksema & Girgus, 1994). These data support the view that depression in adults might

actually originate during the adolescent period (Lewinsohn et al., 1999; Rao et al., 1999).

Depression that occurs during such a critical period of development can lead to impaired

school functioning, disturbed peer and parent relationships and increased rates of health

problems or somatic complaints, as well as long-term serious consequences due to an

increase risk of engaging in high-risk behaviors such as smoking, alcohol and other substance

abuses and even suicide attempts (Asarnow et al., 2005; Bernstein et al., 1997; Deykin et al.,

1992; Egger et al.,1999). Since SSRIs were introduced into the market, due to their safety

profile and benign adverse effects compared to the earlier antidepressants, they have also

been used in the treatment of depression in children and adolescents. However, SSRI side

effects testing has mostly been done in adults; thus their safety in the pediatric population is

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not well-established. In their meta-analyses, Whittington et al., (2004) and Jureidini et al.,

(2004) concluded that the benefits from SSRIs treatment in children and adolescents are

offset by the significant risks of serious adverse events (i.e suicidal behavior), except for FX.

Thus, cognitive-behavioral therapy (CBT) appeared to be a better approach in regard to

efficacy and safety (Harrington et al.,1998). Nevertheless, some subsequent studies have

shown that it is more efficious to treat MDD in adolescent with CBT and FX in combination

compared to either therapy alone.(Goodyer et al., 2007; J. March et al., 2004; March et al.,

2007; Pampallona et al., 2004). However, the availability of CBT is limited in some areas,

thus SSRIs can be prescribed in these circumstances. Currently, only FX and escitalopram

are approved by the FDA for the treatment of depression in adolescents (Hosenbocus &

Chahal, 2011; Ronsley et al., 2010). In Europe, FX is approved by the European Medicine

Agency for children and adolescents with moderate to severe symptoms but only after failure

to respond to 4-6 sessions of psychotherapy (Eaton, 2006). In Canada, Health Canada has not

yet approved any SSRIs for use in children and adolescents because of the concerns about the

adverse effects of these agents, particularly suicidal thoughts and behaviors. Hence, any use

of SSRIs in this population is off-label (Voysey, 2004). Even though FX and escitalopram

were approved for use in the US, the FDA did issue a warning regarding the use of SSRIs in

the treatment of depression in adolescents (2007 > FDA proposes new warnings about

suicidal thinking, behavior in young adults who take antidepressant medications; Public

health advisories (drugs) > suicidality in children and adolescents being treated with

antidepressant medications). Besides the potential increased risk of suicidaity associated

with the use of these agents in children and adolescents, another lethal adverse reaction is the

serotonin syndrome. This syndrome is a relatively rare but lethal condition and is a result of

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overstimulation of peripheral and central serotonergic systems. It can occur at high SSRI

doses, overdose or even at therapeutic dose when combined with other serotonergic

medications (eg: TCA, MAOIs, dextromethorphan, lithium) (Nelson et al., 2007). As

antidepressants are used in patients at high risk for suicide, the risk of intentional overdosing

of the drugs is high. In fact, it was reported that among prescription drug overdoses managed

by poison centers in the US, antidepressant overdoses are the most common (Nelson et al.,

2007). In addition, children less than 6 years of age accounted for 17% of all cases of

inappropriate SSRI ingestion. (Nelson et al., 2007) Since there is still limited information on

adverse effects (other than suicide behaviors) of SSRIs use in children and adolescences,

especially when the drug is used off-label, there is a compelling need for the study of the

safety of SSRIs use in such a special population. Therefore, in this study our aim is to

investigate the acute effect of FX on a variety of physiological, behavioral and endocrine

variables in the lambs at different ages from 10 days to 12 months old.

5.2 Methods:

Animal housing, surgical procedure, experimental protocol and methods are

described in detail in chapter 2.

In this chapter, an additional behavior study was performed in the lambs at 6 months

of age in which lambs were not restricted to the monitoring pen for sample collection.

Instead, lambs were allowed to move freely in the pen with their penmates (peers or siblings)

and have access to food and water ad libitum. Sterile water or FX 1mg/kg were given via

their jugular vein catheter or by venouspuncture around 10am and their behavior were

observed by a continuous DVR system to look for any abnormal behaviors. Through DVR

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observations, their activities were divided into lying down asleep, lying down awake,

standing moving, standing quiet, walking and headbutting. The activities were collected from

the DVR system in 15 second epochs and then matched with the activity score from the

lamb’s actiwatch in the same epoch to provide the intensity for each corresponding activity.

In the lambs from 10 days old onward, cortisol and ACTH measurements in the FX

group were measured in the 3 month and 1 year old groups only.

Statistical analysis:

Differences in the effects of treatment and sex on behavior activities and weight gain

were determined using two-way repeated measure ANOVA. In general, three way ANOVA

was used to determine the effects of treatment, time and sex on cardiovascular-respiratory,

metabolic, endocrine variables. Two-way repeated measured ANOVA or Mann Whitney U

test were used for the non-normally distributed variables to determine the effect of treatment

and time. Delta O2 saturation, which is the difference between O2 saturation at 5 min after

dosing and that at -15 min before sterile water or FX administration, was calculated for each

animal in the control and FX group. Then delta O2 saturation between the control and FX

group was compared using the Mann Whitney U test (in the 1 and 6 months old lambs) or 2

way ANOVA (in the 1 year old lambs). For the data presented as percentage, statistical tests

were run on the raw data, not on % data, except for O2 saturation. P < 0.05 defines significant

differences. Data are presented as mean ± SEM. All data were analyzed using the Sigma Plot

11 statistics software (Systat Software, Inc; San Jose, CA, USA).

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5.3 Results:

Actiwatch activities, arterial pressure, heart rate, heart rate variability, arterial blood

gases (O2 sat, pO2, pCO2, pH, BE, HCO3), lactate, glucose and Hb concentration in FX

experiments in the lambs at all age groups are summarized in the table in the appendices.

5.3.1 Acute FX Effects On Cardiovascular-Respiratory Functions:

There were no significant changes and no gender differences in arterial pressure,

heart rate and HRV variables with acute FX administration in the lambs at 10 days, 1, 3 and 6

months of age even though arterial pressure tended to increase for the first 5 min post-FX in

the lambs at these ages. (data in appendices)

However, in the 1 year old lamb, arterial pressure significantly increased after FX

administration and this effect lasted for about 15-30 mins (Fig. 5.1). Mean arterial pressure

were significantly different between the control and FX group at 1,2,3,4,5, 15 and 30 minutes

post-FX (p<0.001), but not at the control time point (-15 minutes). Pressure increased from

85.1 ± 2.2 mmHg before FX to peak value of 106.6 ± 4.1 mmHg at 2 mins after FX

administration, thus the maximum increase was 21.5 ± 4 mmHg. It is noted that the increase

in mean arterial pressure was more profound and lasted longer in males than in females.

Statistical analysis indicated that the difference in arterial pressure at 2 and 3min after

dosing vs -15min before dosing were only significant in male lambs (p = < 0.001 and p =

0.034). At the 2 min sample, the mean arterial pressure increase was significantly higher in

males than in females (to 112.6 ± 4.1 mmHg in male vs 99.1 ± 5.2 mmHg in female; p =

0.012), with a maximum increase of 25.8 ± 4.4 mmHg in male compared to 16 ± 6.8 mmHg

in female.

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FIG 5.1: Arterial pressure and heart rate changes in 1 year old lambs with acute FX administration a indicates significant differences between variables at -15 min vs 1,2,3,4,5,10,15 min after FX within the FX group b indicates significant differences between control and FX at the corresponding time c indicates significant differences between male and female at the corresponding time within FX group

a a b b b

a a a a a

b b b b b b b

c

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There was a transient decrease in heart rate at 2 min after FX (p = 0.026), the

difference was 30 ± 4 bpm but it quickly returned to normal within 1 min. No sex difference

was found in the heart rate response. (Fig 5.1)

In terms of HRV, we did not find any significant difference between male and female

lambs at 1 year of age. Except for the LF/HF ratio, all other variables (SDNN, SD Delta NN,

RMSSD, LF power (ms2), HF power (ms2) significantly increased at 30 min after FX dosing

in the FX group compared with the control group (SDNN: p = 0.013, SD Delta NN: p =

0.002, RMSSD: p = 0.002; LF power: p = 0.01; HF power: p = 0.002). The difference

persisted for 60 min for some variables (SD Delta NN, RMSSD, HF power). (Fig 5.2)

In 4 male lambs at 1 year of age, delta arterial pressure (i.e the difference between

arterial pressure value at 2 min post-FX and that at -15 min pre-FX) and plasma log FX

concentration at 5 mins followed a linear regression with r2 = 0.84, p = 0.08

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FIG 5.2: HRV (SDNN, SD Delta NN, RMSSD, LF power, HF power, LF/HF ratio) changes with acute FX administration in 1 year old lambs. a indicates significant differences between variables at -30 min vs 30,60,90 and 120 min after FX within the FX group b indicates significant differences between control and FX at the corresponding time

b b

b b

b b

a b

a b b

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5.3.2 Acute FX Effects On Homeostatic And Respiratory Functions:

There was no FX effects on respiratory and metabolic function in the lambs at 10

days and 3 months of age even though Po2 tends to be declined at 5 min post FX in the 3

month-old lambs but this is not significant. (data in appendices)

However, Po2 significantly decreased at 5 min after FX injection in the 1 month-old

lambs (p < 0.001). The FX effect was significantly different between male and female (p =

0.002). In males, Po2 decreased from 92.4 ± 4.4 at 15 min pre-FX to 67.2 ± 4.1 mmHg 5 min

post-FX while in females it fell from 97.6 ± 2.5 at 15 min pre-FX to 92.5 ± 4.5 mmHg 5 min

post-FX. (Fig 5.3) There was also a statistically significant difference in delta O2 sat between

the control group (median = 0.625%) and the FX group (median = -3.35%) (p = 0.031) with

the Mann-Whitney U test. This indicates that FX caused a transient hypoxemia in lambs at 1

month of age and it was more apparent and severe in male than female lambs. Other blood

gas variables (pH, pCO2, HCO3-) and glucose and lactate concentrations were similar between

the control and FX groups in this age group.

Similarly, in the 6 month-old lambs, 2 way (treatment x time) repeated measure

ANOVA revealed a significant decrease of pO2 at 5 min post-FX compared to control (p =

0.035). Po2 fell from 111.9 ± 5.4mmHg at 15 min pre-FX to 95 ± 7.7mmHg at 5 min post-

FX. (Fig 5.4) Delta O2 sat was significantly different between the control group (median =

0.65%) and the FX group (median = -1.05%) (p = 0.004) with Mann-Whitney U test.

However, there were no significant differences in hypoxemia response to acute FX

administration between males and females. Other variables were also not significantly

different between the control and FX groups.

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FIG 5.3: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 1 month of age. a indicates significant differences between -15 min and 5 min after FX within the FX group b indicates significant differences between control and FX within the corresponding time c indicate significant differences between male and female within the corresponding time Asterisk (*) indicates significant differences in delta O2 sat (i.e differences between values at 5 mins and at -15 mins) between control and FX group using Mann-Whitney u test

*

a b c

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FIG 5.4: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 6 months of age. a indicates significant differences between -15 min and 5 min after FX within the FX group b indicates significant differences between control and FX within the corresponding time Asterisk (*) indicates significant differences in delta O2 sat (i.e differences between values at 5 mins and at -15 mins) between control and FX group using Mann-Whitney u test

*

a b

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In the 1 year-old lambs, a transient decrease in Po2 occurred at 5 min after FX

injection and returned to normal by the time of 15min post- FX. Po2 fell from 110.5 ± 2.5

mmHg at 15 min pre-FX to 94 ± 5.8 mmHg at 5 min post-FX. Delta O2 sat was significantly

different between control and FX group and was more severe in male lambs (p = 0.037).

(Fig. 5.5) There was a significant inverse relationship between the changes in Po2 at 5 min

(delta Po2) and changes in arterial pressure at 2 min (delta AP) following FX injection with r2

= 0.54, p = 0.037, n = 8. The greater the increase in arterial pressure at 2 min post-FX, the

greater the decrease in Po2 at 5 min post-FX. (Fig. 5.6A) The significant relationship between

delta Po2 and delta arterial pressure is sex-specific, being significant in males (r2 = 0.93, p =

0.03, n = 4) but not in females (r2 = 0.66, p = 0.18, n = 4) (Fig. 5.6B). Similarly, changes in

O2 saturation (delta O2) at 5 min and delta AP were significantly correlated in male (r2 = 0.98,

p = 0.01, n = 4) but not in female (r2 = 0.27, p = 0.48, n = 4) (Fig. 5.6C). Also, there was a

significant relationship between delta pO2 (i.e. the difference between pO2 at 5 min post-FX

and that at -15 min pre-FX) and log plasma FX concentration at 5 min in lambs at 1 years of

age (r2 = 0.81, p =0.002, n = 8) (Fig. 5.7A). However, the relationship between delta pO2 and

log plama FX concentration at 5 min was not statistically significant in males (r2 = 0.75, p =

0.13, n = 4) and in females (r2 = 0.69, p = 0.16, n = 4) (Fig. 5.7B). Nevertheless, there was a

significant relationship between delta O2 saturation (i.e. the difference between O2 saturation

at 5 min post-FX and that at -15 min pre-FX) and log plasma FX concentration at 5 min in

both males and females with r2 = 0.92, p =0.040, n = 4 and r2 = 0.97, p =0.016, n = 4,

respectively. The higher the plasma FX concentration at 5 min, the greater the decrease in O2

saturation at 5 min following FX injection (Figure 5.7C).

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However, pH value did not change significantly over time and was comparable

between the control and FX group. Pco2 (at -15 min), BE and HCO3 were significantly

different between the control and FX experiments and this occurred only in female lambs

(Fig. 5.8). Similarly, lactate (at 5 min post FX) and Hb (at 5 and 15 min post-FX) were

significantly higher with FX administration compare to placebo only in females (Fig. 5.9).

Glucose concentration was significantly different between male and female lambs in both

control and FX group (Fig. 5.9). There were no significant changes in temperature after FX

administration. (data in appendices)

Arterial blood gas samples were also taken at 2 mins after FX in 5 out of the 10 lambs

in this 1 year age group. Except for pO2 and O2 sat, which were also decreased as shown at 5

mins after FX (Fig. 5.5), there were no significant differences in other blood gas variables at

2 mins after FX compared with those at -15 mins before FX. However, pCO2 tended to

decrease and pH tended to increase at 2 mins post-FX compare with -15 min pre-FX (32.6 ±

1.6 vs 36.8 ± 2.1 mmHg; 7.531 ± 0.027 vs 7.486 ± 0.027, n = 5, respectively) although they

are not statistically significant.

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FIG 5.5: pO2 and O2 saturation with sterile water and FX administration in male and female lambs at 1 year of age. a indicates significant differences between -15 min and 5 min after FX within the FX group b indicates significant differences between control and FX within the corresponding time Asterisk (*) indicates significant differences in delta O2 sat (i.e differences between values at 5 mins and at -15 mins) between control and FX group within male, and between male and female within FX group.

*

a b

A

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FIG 5.6: (A) The relationship between changes in pO2 at 5 mins and changes in arterial pressure (AP) at 2 mins following FX injection in all lambs at 1 year of age (square symbol, solid line, n = 8); (B) The relationship between changes in pO2 at 5 mins and changes in AP at 2 mins following FX injection in male (square symbols, dotted line, n = 4) and female (circle symbols, solid line, n = 4) at 1 year of age and (C) The relationship between changes in O2 saturation at 5 mins and changes in AP at 2 mins following FX injection in male (square symbols, dotted line, n = 4) and female (circle symbols, solid line, n = 4) at 1 year of age.

A

B

C

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FIG 5.7: (A) The relationship between changes in pO2 at 5 mins and log FX concentration at 5mins following FX injection in all lambs at 1 year of age (square symbol, solid line, n = 8) (B) The relationship between changes in pO2 at 5 mins and log FX concentration at 5mins following FX injection in male (triangle symbols, dotted line, n= 4) and female (circle symbols, solid line, n = 4) lambs at 1 year of age; and (C) The relationship between changes in O2 saturation at 5 min and log FX concentration at 5mins following FX injection in male (triangle symbols, dotted line, n= 4) and female (circle symbol, solid line, n = 4) lambs at 1 year of age.

A

B

C

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FIG 5.8: PCO2 , base excess (BE), HCO3 concentrations with sterile water and FX administration in male and female lambs at 1 year of age.

140

FIG 5.9: Glucose, lactate, Hb (Hemoglobulin) concentrations with sterile water and FX administration in male and female lambs at 1 year of age.

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5.3.3 Acute FX Effects On Behavior:

There were no differences in activity variables between the average of 3 normal days,

control group and the FX group in the ~ 10 day-old group. In contrast, activities significantly

decreased on the FX experimental day compare to both control experimental day and normal

day in the 1 month-old lambs. Starting from 3 months old, the lambs were kept in the

monitoring pen for 4 days during the control and FX experiments. Thus, their activities

decreased on both control and FX experiment day due mainly to the restraint. Summarized

data for all age groups are in tables 1, 2 in the appendices. To investigate the actual FX

effects on the older lambs (after 3 months old) we conducted a behavior study on the 6

month-old lambs in which they were housed in their regular holding pen with their sibling or

peers as usual, as described in the method section. The activity data of this behavior study is

presented in figures 5.15-5.17. Note that activity data of the 6 month-old group presented in

the appendices are the activities when the lambs were kept in the monitoring pen.

5.3.3.1 Behavioral Activities In The Lambs At 1 Month Of Age:

Active bouts per day, mean score in active and total activity score were significantly

lower on FX experimental day as compared to the average of 4 normal days (p<0.001) while

they were comparable between the other days (control experiment day, post FX day 1, post

FX day 2) and the average of 4 days. In contrast, episode duration was significantly higher on

the FX day compared to the average of the 4 normal days. Even though the percentage of

moving time was lower and the percentage of time immobile were higher on the FX day

compared to the average of 4 normal days, respectively, they were not statistically

significant. (Fig 5.10) There were no sex differences in these actiwatch variables.

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Results from DVR observations showed that the intensity of suckling attempts

determined by actigraphy was significantly higher on the FX day compared to the normal day

(FX day: 327 ± 21 counts/15s versus normal day: 247 ± 28 counts/15s). The lambs always

attempted to suckle when they were returned to their mothers after every blood sampling but

the ewes almost always refused them. Thus, it is reasonable that the intensity of suckling

attempts was higher on the FX days as lambs tried harder. Importantly, there were no

significant changes in the percentage of time lamb spent on suckling attempts on the FX days

compared to normal days, which indicates that blood sampling did not interfere with their

feeding behavior. However, the amount of time lambs spent on successful suckling was

significantly lower on the day of the FX experiment (FX day: 0.7 ± 0.14 %/24h versus the

normal day: 1.1 ± 0.22 %/24h). In addition, the amount of time that lambs spent walking

significantly decreased on the FX experiment day (3.6 ± 0.2%/24h) vs the control day (4.9 ±

0.4 %/24h) (Fig 5.11A). Even though the time lambs spent standing was not significantly

different between the days, when dividing the standing activity into standing quiet (defined

by standing from DVR observation and actiwatch score less than 60) and standing mobile

(defined by standing activity from DVR observation and actiwatch score equal or greater

than 60), there was a significant decrease in the amount of time lambs spent standing moving

on the FX experiment day (8.9 ± 0.9 %/24h) compared to the control experiment day (13.7

±1.5 %/24h) or normal day (13.7 ± 1.3 %/24h), (p = 0.013). Moreover, the intensity of

standing quiet was also significantly lower on the day of FX experiment (28 ± 2.8

counts/15s) versus control day (34 ± 2.3 counts/15s) (Fig 5.12). Although the time lambs

spent for experiments was significantly higher on the control and FX day compared to

normal day, it was negated by the significantly lower intensity of activity during the

143

experiment on experiment days compared to normal days (Fig 5.11). In fact, lambs slept or

lay awake most of the time during the experiments. Collectively, the results suggest that 1

month-old lambs were less successful at suckling and less active (walking less, moving less

and more still while standing) with FX administration. However, their weight gain was

comparable between the average of normal days, control, FX experiment and post FX days.

(Fig 5.13)

Regardless of the day, there were a sex difference in the intensity of standing and

playing activity. Female lambs had a higher average score of standing activity (normal day:

102 ± 2.2, control day: 110 ± 11, FX day: 93 ± 16 counts/15s) than the male (normal day: 74

± 6.6, control day: 75 ± 8.2, FX day: 63 ± 3 counts/15s). Similarly, there was higher playing

activity in females than in males (normal day: 272 ± 33 vs 178 ± 44; control day: 232 ± 43 vs

164 ± 18, FX day: 265 ± 15 vs 174 ± 31 counts/day) (Fig 5.14). In general, around 1 month

of age female lambs were more active than male lambs but this was independent of FX

administration.

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FIG. 5.10: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in 1 month-old lambs with the average of the 4 normal days in the pen before the experiments (n = 12, open bar), on control experiment day (n=9, black bar), and on FX experiment day (n = 12, dark gray), post FX day 1 (n = 11, light gray), post FX day 2 (n = 11) and post FX day 3 (n = 11). (*) indicates statistically significant between FX experiment day and average of 4 days, p< 0.05.

145

FIG. 5.11: (A) Percentage of time lambs spent for each activity from DVR observation and (B) the intensity of each activity when matching DVR observation with actiwatch score data in lambs at 1 month of age. Normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). (^) indicates significant differences between control experiment day and normal day (+) indicates significant differences between FX experiment day and control experiment day (*) indicates significant differences between FX experiment day and normal day, p < 0.05.

A

B

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FIG. 5.12: (A) Percentage of time lambs spent for standing activity from DVR observation and (B) the intensity of standing quiet and standing moving when matching DVR observation with actiwatch score data in lambs at 1 month of age. Normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). (^) indicates significant differences between control experiment day and normal day (+) indicates significant differences between FX experiment day and control experiment day (*) indicates significant differences between FX experiment day and normal day, p < 0.05.

A

B

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FIG 5.13: Weight gain (mean ± SE) in the lambs during their normal days, control experiment with distilled water injection, FX experiment with FX injection and 3 continuous days after the FX experiment day around 1 month of age.

FIG. 5.14: Intensity of standing and playing activity from DVR observation in the 1 month-old male and female lambs on normal day (day lambs in the pen without any experiments, gray bar, n = 3), on Control experiment day (n=6, open bar) and FX experiment day (n=6, black bar). (a) indicates significant differences in standing activity on each corresponding day between male and female. (b) indicates significant differences in playing activity on each corresponding day between male and female. p < 0.05.

148

5.3.3.2 Behavioral Activities In The Lambs At 6 Months Of Age:

Active bouts per day, total activity score and % time moving were significantly lower

on the FX experimental day and post FX day 1 as compared to the average of 4 normal days

(p = 0.012, p = 0.007, respectively) while they were comparable between the other days

(control experiment day and post FX day 2) and the average of 4 days (Fig. 5.15). In contrast,

% time immobile was higher on the FX day and post-FX day 1 compared to the average of 4

normal days. Mean score in active tends to be lower and episode duration higher on the day

of FX experiment but these changes were not statistically significant. (Fig 5.15) There were

no sex differences in these activity variables from the actiwatch measurements.

The results from the DVR observations showed that there was a small but significant

increase in the amount of time lambs spent on headbutting on the day of the FX experiment

(0.8 ± 0.17 %/24h) compared to the day of control experiment (0.39 ± 0.09 %/24h) (p =

0.036) but not significant to the normal day (0.47 ± 0.09 %/24h) (Fig 5.16A). Even though

the amount of time spent for standing was not significantly different between the days, when

dividing the standing activity into standing quiet (defined by standing from DVR observation

and actiwatch score less than 60) and standing mobile (defined by standing activity from

DVR observation and actiwatch score equal or greater than 60), there was a significant

decrease in the amount of time lambs spent for standing moving (FX: 14.4 ± 1.51%/24h,

control: 21.2 ± 2.9 %/24h; normal: 21.2 ± 3.06 %/24h) and an increase in the time standing

quiet (FX: 18.2 ± 1.53 %/24h, control: 12.2 ± 1.44 %/24h, normal: 13.6 ± 2.02 %/24h) on the

FX experiment day compared to the control experiment day or normal day (p = 0.027 and p =

0.005, respectively) (Fig. 5.17). In addition, the intensity of standing activity was

significantly lower on the day of FX experiment (68 ± 7.2 counts/15s) compare to the normal

149

day (102 ± 15 counts/15s) (p = 0.042) (Fig. 5.17B) and this difference was due to the

significant differences in the intensity of standing quiet (FX day: 12 ± 1.4 counts/15s, control

day: 18 ± 1.9 counts/15s, normal day: 18.9 ± 1.9 counts/15s) (Fig 5.17). These results

suggested that after FX administration the lambs’ behavior were shifted toward two extremes

of activity: they were less active, stood very still for longer time, but were also more

aggressive indicated by an increase in headbutting activity. It was noted from DVR

observations that two lambs (one male and one female) presented with jerky movements

from around 20.5h after FX administration. These jerky movements occurred intermittently

in the lambs for ~10h during the day. However, no differences in the intensity and the % time

of each activity were found between male and female lambs. Unfortunately, no blood

samples were available for the measurement of FX concentration in these lambs.

Interestingly, at 6 months of age, acute FX administration resulted in a significant

weight loss in the lambs (-0.7 ± 0.19 kg, n = 8) as compared to an average of daily weight

gain of 0.1 ± 0.05 kg at their age (p = 0.011). (Fig 5.18) This FX effect on bodyweight gain

was not observed in the younger lambs.

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FIG. 5.15: Activity variables (active bouts per day, mean score in active, episode duration, total activity score, % time moving, % time immobile) in 6 month-old lambs with average of 4 normal days in the pen before the experiments (n = 8, open bar), on control experiment day (n=8, black bar), and on FX experiment day (n = 8, dark gray bar), post FX day 1 (n = 8), post FX day 2 (n = 8) and post FX day 3 (n = 8). Asterisk (*) indicates statistically significant between FX day or post FX day 1 and average of 4 normal days, p< 0.05.

151

FIG. 5.16: (A) Percentage of time lambs spent for each activity from DVR observation and (B) the intensity of each activity when matching DVR observation with actiwatch score data in lambs at 6 month of age. The normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). (+) indicates significant differences between FX experiment day and control experiment day (*) indicates significant differences between FX experiment day and normal day, p < 0.05.

A

B

152

FIG. 5.17: (A) Percentage of time lambs spent on standing (quiet and moving) activity from DVR observation and (B) the intensity of standing quiet and standing moving when matching DVR observation with actiwatch score data in lambs at 6 month of age. The normal day was a day lambs were in the pen without either control or FX experiment (n = 6, gray bar). Control experiment (n=6, open bar), FX experiment (n=6, black bar). (+) indicates significant differences between FX experiment day and control experiment day (*) indicates significant differences between FX experiment day and normal day, p < 0.05.

A

B

153

FIG 5.18: Weight gain (mean ± SE) in the lambs during their normal days, control experiment with distilled water injection, FX experiment with FX injection and 2 continuous days after the FX experiment day at around 6 months of age. (*) indicates significant differences between FX experiment day and the normal average of 4 days, p < 0.05.

5.3.3.3 Acute FX Effects On Cortisol Level:

No differences were found between the cortisol concentrations in the control group

compared with the FX group at 3 month and 1 year old, nor ACTH concentration. (Figs 5.19

& 5.20)

There were also no differences in cortisol level between sexes.

154

FIG. 5.19: Cortisol and ACTH concentration in the lambs at 3 months of age at 15, 30 minutes before and 0.5, 1, 2, 6 and 12 h after sterile water injection (control group) and FX injection ( FX group)

155

FIG. 5.20: Cortisol and ACTH concentration in the lambs at 1 year of age at 15, 30 minutes before and 0.5, 1, 2, 6 and 12 h after sterile water injection (control group) and FX injection (FX group)

156

5.4 Discussion:

In this study which included lambs at 10 days, 1, 3, 6 month and 1 year of age, we

found that acute i.v. FX administration did not have any significant effects in the lambs at 10

days and 3 months of age. However, transient hypoxemia occurred in the 1 month old lambs

with acute FX administration and it was more profound in males. Lambs were also less active

and altered their feeding behavior on the day after FX administration. These FX effects on

respiration and behavior were also observed in the lambs at 6 months of age but there were

no sex differences. At 1 year of age, acute FX administration resulted in an increase in

arterial pressure, to a greater extent in the male than the female, and a transient decrease in

heart rate. HRV was also altered with FX administration for about 30-60 minutes in both

sexes. No changes in cortisol level occurred with acute FX administration.

The daily therapeutic FX dosing for children from 6 to 18 years old ranges from 10 to

20 mg but no mg/kg dose has been provided (Eaton, 2006). However, according to a

reference used by poison control centers in the US, a minimum toxic dose of FX in children

less than 6 years of age is 60 mg or 5mg/kg (Nelson et al., 2007). In our study 1mg/kg FX

dose was used, which is at the high margin of the therapeutic range but does not reach a

minimal toxic dose. Moreover, the plasma FX concentrations achieved in the lamb

experiments were in the therapeutic FX concentration range in humans (Chow, 2013).

In our study, increase in arterial pressure and decrease in heart rate after acute FX

administration were observed in the older lambs. As the mechanism of SSRI group, plasma

5-HT concentration is expected to increase after FX bolus by the inhibition of 5-HT reuptake

by platelets, the main reservoir of peripheral 5-HT (Maurer-Spurej et al., 2004; Ortiz &

157

Artigas, 1992). 5-HT has been known as a vasoconstrictor agent, its increase would lead to

an elevated blood pressure (Maurer-Spurej et al., 2004). Even though FX seems to have

divergent effects on blood pressure, (Lazartigues et al., 2000; Tsai & Lin, 1986) did report

that central acute administration of FX caused hypertension in rats in a dose-dependent

fashion. In humans FX is usually administered for longer time durations (several weeks) per

os and an increase in blood pressure was also observed in those subjects who had a normal

pretreatment mean diastolic pressure (Amsterdam et al., 1999). The diversity of 5-HT effects

on blood pressure in the literature could be attributed to many factors such as differences in

species, route of administration, doses, and 5-HT receptor subtypes. However, elevated

arterial pressure after acute FX injection in the older lambs in our study was consistent with a

report by (Nelson et al., 1987) in which increase in blood pressure was observed with 5-HT

injection in conscious sheep.

However, our study is probably one of the first to examine blood pressure responses

to FX administration over age in relation to gender. The effect of FX on arterial pressure

elevation only became statistically significant in lambs at 1 year of age and it also increased

higher in males compared to females. It is not yet clear why the FX effects on blood pressure

was only significant at 1 year of age. However, one explanation could be due to either a

lower level of platelets, which are the main reservoir of peripheral 5-HT, in the younger

lambs or a lesser extent of a functioning 5-HT receptor subtypes that mediate 5-HT effects.

Platelet concentrations are lower in human newborns and children compared to adults

(Merritt & Davidson, 1933) and in addition, the platelet 5-HT concentration in newborns is

only ~42% of the value in children and adults (Flachaire et al., 1990). If a similar situation

occurs in sheep, the transient increase in blood 5-HT following SSRI administration may

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have been less in the younger lambs and not sufficient to elicit effects on arterial pressure and

blood oxygenation. Measurement of platelet concentrations, platelet 5-HT concentration and

the magnitude of the transient increase in blood 5-HT concentration following SSRI

administration in sheep as a function of age could confirm this hypothesis.

In terms of the sex differences in the FX effects on arterial pressure in the year old

lambs, the lung is a major site of clearance of plasma 5-HT, with ~95% of 5-HT being

removed from plasma by the lung via a first-pass effect (Gillis & Pitt, 1982). However, a

small change in the 5-HT clearance rate by the lung can dramatically affect of the systemic

levels of 5-HT, for example increasing pulmonary extraction from 95 to 97.5% would reduce

plasma 5-HT concentration in the systemic circulation by 50%. (Gillis & Pitt, 1982) Estrogen

(17ß estradiol) or progesterone appears to increase 5-HT metabolism in lung homogenates

prepared from female rats (Bakhle & Ben-Harari, 1979). By one year of age, male and

female lambs who are raised indoors under the same condition as ours have undergone

puberty (Suttie et al., 1991) and the females have thus initiated estrous cycles (Ryan et al.,

1991; see Chapter 3), with pulsatile increases in blood estrogen and progesterone levels.

Thus it is possible that an effect of ovarian steroids on increasing 5-HT clearance by the lung

is responsible for the observed smaller increase in the arterial pressure response to FX

administration in the 1 year old female lambs compared to male lambs, because of a lesser

transient increase in blood 5-HT levels following FX injection. It has been reported that the

rate of sustained hypertension is higher in adults who underwent 12 weeks of venlafaxine (a

serotonin and noradrenaline reuptake inhibitor) treatment for depression compare to placebo

group (Feighner, 1995). In general, SSRIs are not considered to be cardiotoxic, in

comparison to the classical antidepressants (TCA, MAOI) (Lane et al., 1995). However,

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hypertension is one of the symptoms included in serotonin syndrome (Boyer & Shannon,

2005) and the serotonin syndrome has been reported in pediatric and adolescent populations

after SSRI administration (Asch & Parker, 1988; Gill et al., 1999). However, we did not

observe hyperthermia after FX injection in the lambs in our study (data in appendices), and

hyperthermia is also a symptom of the serotonin syndrome (Boyer & Shannon, 2005).

In the 1 year old male lambs the magnitude of increase in arterial pressure at 2 min

following FX injection (i.e: peak value) and plasma FX concentration at 5 mins followed a

linear regression analysis. Even though the relationship was not statistically significant (p =

0.08) because of the small sample size (n = 4), it to some extent implies a correlation

between FX concentration and its effect.

Interestingly, we found HRV variables (SDNN, LF power, SD Delta NN, RMSSD,

HF power) increased for the first 30 mins after acute FX administration in the older lambs. In

the literature, the effects of SSRIs on HRV are still a matter of debate. It has been established

that HRV decreases in depressed patients, which increases the risk of cardiovascular disease

and mortality in these patients (Carney et al., 2005; Grippo & Johnson, 2002; Thayer &

Lane, 2007). It has also been shown that TCA treatment exacerbated the decreased HRV in

depressed patient which could contribute to the adverse cardiovascular side effect of TCA

(van Zyl et al., 2008). However, there is more controversy when it comes to SSRIs effect on

HRV. Recent publications reported an “unfavorable” decrease (Licht et al., 2008; Licht et

al.,2010) or unchanged of HRV (Kemp et al., 2010) with SSRIs treatment in depressed

patients. However, in a meta-analysis done by van Zyl et al., (2008), SSRI use was

associated with a slight decrease in heart rate and increase in SDNN. Among SSRIs included

in this meta-analysis, FX was investigated in only one study by Khaykin et al, which reported

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an increase of SDNN (Khaykin et al., 1998; van Zyl et al., 2008). This finding is consistent

with our HRV results, at least in terms of the SDNN variable. In addition, we did observe

increases in other HRV variables (SD Delta NN, RMSSD, HF power, LF power) after acute

FX administration. However, in the present study, it is unclear why both LF power and HF

power increase at the same time. One possibility is that FX might increase sympathetic tone

but, in parallel, increase vagal tone and thus minimize the heart rate changes.

However, the significant increase in HRV was only observed in the lambs at 1 year of

age and not in the younger lambs. Given the developmental aspects of the lambs, they were

deemed to be an adult at 1 year old. Therefore our results suggested the HRV response to

acute FX is age-dependent in parallel with the maturation of the autonomic system and

adolescents might respond differently to the drug as compared to the adult.

In vivo studies have shown an opposite respiratory response to acute and chronic

SSRI treatment. While acute SSRIs (FX, PX) administration caused a reduction in respiratory

rate and minute volume in alert, freely moving rats; chronic treatment for more than 23 days

in those rats led to an increase in respiratory rate (Annerbrink et al., 2010). In our study, we

did not record respiratory rate, instead the effect of FX on respiratory system and

homeostasis was observed through the changes in the lambs’ arterial blood gas status. We

found a decline in pO2 and O2 saturation in the lambs at 1, and 6 months and 1 year of age at

5 mins after FX administration. However, there were no significant accompanying changes in

pCO2, pH, HCO3- and lactate concentration after FX. FX and other SSRIs agents when given

acutely will lead to an acute increase in synaptic levels of serotonin by blocking serotonin

reuptake (Stahl, 1998). There is evidence to suggest that serotonin might exert both

stimulatory and inhibitory influences on respiration. (Annerbrink et al., 2003; Mulkey et al.,

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2004) Thus the observed reduction in pO2 and O2 sat after FX administration could be a result

of respiratory inhibition due to an increase in synaptic serotonin concentrations. Although

due to technical difficulties we did not succeed in an attempt to measure plasma serotonin

following FX administration in the lambs in our study, a study in rat has shown that after

acute SSRI (FX) administration plasma serotonin increased 520% as compared to the control

(Ortiz & Artigas, 1992). The increase in serotonin also occurred in previous study in our lab

in which plasma serotonin level increase for the first hour after the bolus of loading FX dose

in 3 adult non-pregnant ewes (Morrison, 2001). In addition, in the present study, we found

that there was a significant relationship between the level of hypoxemia at 5 mins post-FX

and the magnitude of increase in plasma FX concentration at 5 mins in both male and female

lambs at 1 year of age. The higher the level of FX, the greater the decrease in O2 sat or

hypoxemia in the 1 year old lambs. This again confirms the direct effects of FX on

physiological changes. As it was mentioned above, a study in rats showed that the effect of

acute FX on reduced respiratory rate only occurred at the dose of 10mg/kg to achieve mean

plasma FX = 1092.8 ± 315.9mmol/L, NFX = 1203.2 ± 308.2 mmol/L and a more reduced

respiratory rate at a higher dose of 30mg/kg while there was no FX effect at the dose of 1

mg/kg. (Annerbrink et al., 2010) Similarly, in a human study, SSRI effects on fetal behavior

were only observed in the fetuses exposed in utero to the drug at standard and high doses in a

dose-dependent manner but not in the low dose group (Mulder et al., 2011). Roca et al.,

(2011) also reported an increased risk of preterm birth mainly occurred in women treated

with high dose SSRIs during pregnancy.

Another possibility is that hypoxemia occurred as a result of acute increase in

serotonin which caused vasoconstriction and sudden increase in blood pressure. This could

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result in a decrease in cardiac output due to an afterload effect, which would reduce

pulmonary blood flow to result in the decrease in arterial pO2. In fact, our data have shown

that the magnitude of increase in arterial pressure at 2 min following FX injection was

significantly related to the magnitude of hypoxemia reflected by a decrease in Po2 at 5 min

following FX in the 1 year old lambs supporting for this hypothesis. The relationship was

stronger in males than in females. In addition, an experimental study in dogs showed that

pulmonary arterial pressure increased after 20-30 mins of i.v serotonin infusion (Sackner,

Will, & DuBois, 1966), which suggests pulmonary vasoconstriction. The decrease in pO2

could via chemoreceptor activation lead to hyperventilation leading to a decrease in arterial

pCO2 and increase in pH. This is consistent with the trend of pCO2 and pH changes observed

at the 2 min sample in the 1 year old lambs. However, we did not record respiratory rate and

so could not determine if in fact hyperventilation did occur, and thus explain the unchanged

pCO2 and pH at the 5 min sample.

It is unclear why hypoxemia after FX injection only occurred in the lambs at 1, 6, 12

months of age but not at the other ages (3, 10 days and 3 months of age) and why it was more

severe in male than female at 1 month of age. However, it appeared that FX-induced

hypoxemia was coincident with milestone events in their development (i.e weaning, puberty).

Other factor(s) which are associated with these events might contribute to the observed FX

effects on respiratory and homeostasis in the lambs.

Behavior studies in which lambs were not restrained to the monitoring pen were

performed in lambs at 3, 10 days and 1, 6 months of age. In general, at 1 and 6 months of

age, lambs were significantly less active on the day after FX administration as shown from

the actiwatch data (Figs 5.9 & 5.14). The reduction in their overall activity could be due to

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either the fall in the intensity of high score activities (such as standing moving, walking,

suckling and playing or headbutting) and/or a reduction in the time they spent for these high

score activities over the day. Using a combination of DVR observations and actiwatch score

data, we found that the reduction in the 1 month old lambs after FX injection was a result of

both the reduction in the intensity of standing quiet and a decline in the time lambs spent for

standing moving, walking and successful suckling. One question is whether this reduction in

activity following FX administration at 1 month has any relation to the normal reduction in

lambs’ activity observed around 1 month of age due to weaning onset. Our developmental

study in chapter 3 showed that the 1h rhythm due to suckling is a prominent rhythm in lambs

for the first month of life. That means their main state for this first month of life was simply

either sleeping or suckling. Our results suggested acute FX administration did not disrupt

their feeding activity before 1 month since there were no significant differences in activities

in experiments at 4 and 10 days of age. In parallel of the disappearance of the 1h rhythm

around 1 month, a circadian rhythm and other ultradian rhythms (8h, 6h) emerged and

became dominant (Fig 3.14). Since when this 1h rhythm disappeared, their total activity also

decreased, it is tempting to postulate that this led to a decrease in activity in the FX

experiments at 1 month. If this is the case, one would expect to see the same level of activity

on the days after FX compared to that on the day of FX experiment. However, based on the

pattern of decreased activity illustrated in Fig 5.8, this is less likely the case since the activity

dropped sharply on the day of FX compared to the normal day and recovered quickly right on

the next day after FX back to their previous higher level of activity and remain at that level

days after. In addition, given the fact that lambs were disturbed more by blood sample

collection on the day of control/FX experiment, their activity might have been expected to be

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higher on those days compared to normal days rather than exhibiting a decrease in activity.

However, the reduction in the lamb activity did not affect weight gain, probably because

lambs at this age have already started to consume solid food and although they were less

successful suckling on the day of FX experiment, they did attempt to suckle harder. Taken

together, the effect of FX on the lambs’ activity at 1 month of age does not appear to be

simply due to weaning although it could be a predisposing factor. Instead, the mechanism for

the decreased locomotor activity in the lambs might be located at the neurotransmitter level.

However, since FX has an antidepressant effect, the finding that activity decreased with FX

administration was unexpected. Nevertheless, in clinical practice, it usually requires at least

two weeks of treatment before FX can exert its antidepressant effect (Stahl, 1998). Thus,

acute administration could elicit a paradoxical decrease in activity through an activation of 5-

HT autoreceptor which causes an initial silencing of serotonergic neuron activity (Stahl,

1998). In addition, in our study FX was administered to healthy lambs, which might result in

different effects on serotonergic tone compared to individuals with depressive symptoms.

At 6 months of age, a decrease in the lamb activity was a result of a shift in the time

lambs spent standing moving to standing quiet and a reduction in the intensity of standing

quiet activity. However, the FX exposed lambs were headbutting more on the day of FX

experiment. That suggests that these lambs were quieter but also more aggressive with acute

FX administration. In addition, we also observed jerky movements, which could represent for

one of the symptoms of serotonin syndrome (eg: tremor or akathisia) (Boyer & Shannon,

2005), in two of the eight lambs in this study. Blood samples for FX measurement were not

available for those lambs but it is possible that the effects of FX on the lambs’ behavior was

due to an acute increase in 5-HT available at the synapse on serotonergic neurons which

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regulate the affective behaviors and motor tone (Boyer & Shannon, 2005). Interestingly, the

decrease in activity following acute FX administration in the lambs at 6 months of age was

associated with a bodyweight loss. As a vast majority of their usual standing moving at this

age is attributed to food intake activity at fixed and ad libitum meals, their weight loss

appeared to be consistent with DVR observations showing a decline in the time they spent

with standing moving after FX injection. However, whether the FX-induced decrease in

activity interfered with eating and led to a loss in their body weight or alternatively whether

FX caused a reduction in pre-meal appetite and food intake which in turn led to a decrease in

activity remains unclear. Evidence from human and rodent studies have shown that FX has

an inhibitory role on feeding behavior. (Clifton et al,. 1989; Lawton et al,.1995). Elevated

serotonin at the synapse following FX administration might interact with other neuropeptide

systems (neuropeptide Y, orexin, melanocortin) to alter feeding behaviors (Halford et

al,.2007).

Few studies have examined the HPA axis response following acute FX

administration. In rats, acute FX administration caused an increase in CRH, AVP, ACTH and

corticosterone (Fuller et al., 1976; Fuller et al., 1996; Gibbs & Vale, 1983). Similarly,

cortisol level increased following i.v infusion of citalopram (Bhagwagar et al., 2002) or after

a single dose of FX (von Bardeleben et al., 1989) in healthy humans. In sheep, Broadbear et

al., (2004) found an increase in ACTH and cortisol for 40 mins following acute subcutaneous

sertraline injection in gonadectomized ewes. However, we did not observe any significant

changes in ACTH and cortisol in lambs at 3 months and 1 year of age following acute FX

administration. The differences in our result and the Broadbear et al study could be due to the

route of administration and the sampling period. FX was given intravenously and the earliest

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sample of ACTH and cortisol after dosing was at 30 mins in our study while sertraline was

given subcutaneously and blood was sampled every 10 mins in the other study. In addition,

sex hormones could be a contributing factor in different results since in the subsequent study,

Broadbear et al.,(2004) did show that the increase in ACTH and cortisol in gonadectomized

sheep was abolished by sex hormone treatment. Moreover, it is believed that the weak

response of the HPA axis to acute SSRI administration is due to the activation of the negative

feedback on the 5-HT1A autoreceptors (Raap & Van de Kar, 1999). The midbrain

serotonergic system and HPA axis are highly interrelated; this is mediated by 5-HT1A

receptors. (Porter et al., 2004) An overactive HPA axis which is not suppressed by

dexamethasone administration is a potential long-term suicide predictor (Pompili et al.,

2010). However, whether the finding that no changes in basal cortisol and ACTH level after

acute FX administration in our lambs can indirectly imply that FX does not have effects on

suicidal behavior in the adolescent humans is uncertain.

In conclusion, acute FX effects on cardiovascular-respiratory function and behavior

were seen in some, but not all age groups. The effect tended to occur at milestones events of

development such as weaning, puberty and adulthood. Gender difference in FX effect on the

cardiovascular-respiratory function was observed in the older lambs with more profound FX

effects in males.

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6. Postnatal Outcomes in Lambs Exposed Antenatally to FX

6.1 Introduction:

Up to 20% of pregnant women might experience mood disturbances at any stage of

their pregnancy and thus might require antidepressant therapy (Burt & Stein, 2002). SSRIs

have been widely used as a pharmacological treatment for depression during pregnancy with

moderate to severe symptoms thanks to their relatively benign side effects profile compared

to other earlier antidepressants (MAOIs, TCA). Indeed, over the last decade, SSRI use during

pregnancy has increased from 1.5% in 1996 to 6.4% in 2004 (Andrade et al., 2008). A

similar increase to approximately 7% was also reported by Wichman et al., (2008). As the

name suggests, SSRIs exert their actions by inhibiting serotonin recycling through the

serotonin transporter, thereby increasing the amount of serotonin available in the synaptic

cleft, and altering serotonergic transmission (Oberlander et al., 2009). It has been well-

established that in adults serotonin is a neurotransmitter which plays a key role in modulating

emotion, cognition, attention, arousal and stress responses. In the developing brain, central

serotonin also acts as a growth factor regulating serotonergic neuronal growth and the

development of other related neural systems (Whitaker-Azmitia, 2001). Since SSRIs are

capable of crossing the placenta (Hendrick et al., 2003; Kim et al., 2004; Kim et al., 2006;

Rampono et al., 2009), there is a significant risk for the fetus from exposure to SSRIs during

such critical phases of their neurodevelopment. Studies in human and sheep have

demonstrated SSRIs effects on the fetuses. Mulder et al reported a disrupted quiet sleep

reflected by the abnormal phenomenon of continual bodily activity during non-REM sleep

periods in human fetuses in utero exposed to SSRIs (Mulder et al., 2011). Similarly,

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Salisbury et al (2009) demonstrated increased jerky movements and decreased fetal breathing

movements in SSRIs-exposed fetuses at 26 and 36 weeks gestational age as compared to the

non exposed group. This is in agreement with previous findings from our group which

showed an alteration of fetal behavioral state and neuroendocrine function in fetal lambs with

chronic FX infusion to the mothers during late gestation (Morrison et al., 2001; Morrison,

2001; Morrison et al., 2004). Moreover, a recent study also reported an alteration of fetal

middle cerebral artery flow characteristics and blunted fetal heart rate variability across the

day in 36 week human fetuses exposed to SSRIs as compared to the control group (Rurak et

al., 2011). These findings raise an important question whether these fetal neurobehavioral

effects persist beyond the perinatal period into childhood, forming a concept of behavioral

teratology in the neonate resulting from fetal exposure to SSRIs. A blunted pain response in

terms of facial characteristics and cardiac responses in prenatally SRIs-exposed neonates has

been reported by Oberlander et al., (2002). This alteration in pain reactivity was still apparent

at 2 months of age in the fetal SSRIs-exposed infants, even after controlling for drug level

and maternal mood (Oberlander et al., 2005). In addition, a lower basal cortisol level in the

evening was also observed at 3 month of age in prenatal SSRIs-exposed infants (Oberlander

et al., 2008)

Yet, it is still inconclusive regarding the long-term effects of prenatal SSRI exposure

on neurobehavioral development in early childhood. Some studies showed a delayed in

psychomotor and behavioral development in prenatally SSRIs-exposed children (Casper et

al., 2003; Pedersen et al., 2010) while others failed to observe these differences (Heikkinen et

al.,2002; Heikkinen et al.,2003; Nulman et al., 1997; Nulman et al., 2002). Nevertheless, a

challenge for this type of research in human is that it is impossible to distinguish between the

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effects of SSRIs and the effects of maternal psychiatric illness on the outcomes. Moreover,

the postnatal social environment of a depressed mother could also have negative influences

on child development after SSRI exposure (Oberlander et al., 2008). These issues might be

resolved with animal studies. Since lambs are precocious at birth and have a very similar

physiological characteristics to the human, it appears to be a good model to control for the

confounding factors present in human studies. In this study, we chose to investigate the

postnatal outcomes including birth weight, postnatal weight gain, behavioral development

(neonatal behavior, sleep-rest cycles/activities, feeding activity) and cardiovascular changes

(heart rate, HRV, sPO2) in lambs exposed to FX during late gestation.

6.2 Methods:

6.2.1 Animal And Catheter Implantation:

Seven time-bred pregnant ewes (Dorset/Suffolk Cross) were bred following the same

protocol in our laboratory described previously in Chapter 2. Ewes were moved to Centre of

Comparative Medicine (CCM), UBC 2-3 weeks before their due date (term = 147 days) to

allow for acclimation.

They were housed in groups of two or three in the holding pens of approximately

10x12 feet and were able to go outdoors through a small door in their pen until the end of

their pregnancy. Approaching their due date, ewes were kept indoors under natural light

conditions. Ewes were fed following the protocol in the CCM: 300 g grain + 450 g alfalfa

cubes at 7:00 to 8:00 am, 2 flakes of hay at 8:00 to 9:00 am and 2 flakes of hay at 2:00 pm.

The study involved 2 groups of lambs: one group of 7 lambs who were exposed to FX

antenatally and another control group of 2 lambs born in CCM and 10 control lambs

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previously born in the CFRI without any perinatal FX exposure for the first 11 days after

birth.

A heparin-bonded 5.0 French polyurethane catheter (Instech Solomon, Plymouth

Meeting, PA) was percutaneously implanted to the ewe’s jugular vein using a 13G sterile

stainless steel needle (Instech Solomon, Plymouth Meeting, PA) under local analgesia with

Lidocaine HCl 2% (Bimeda-MTC, Animal Health Inc, Cambridge, Ontario). The catheter

was implanted at an average gestational age of 131 ± 1 d in 5 pregnant ewes. The catheter

was inserted approximately 20cm and proper functioning of the catheter was ensured before

it was secured to the skin by a 2-0 Nylon suture. The external part of the catheter was closed

with a sterile metal pin at the end and kept in a small ziplock bag and safety pinned under a

bandage around the animal’s neck. The catheters were flushed every day with heparinized

saline and served for subsequent daily FX injection and blood samplings at delivery.

6.2.2 Experimental Protocol:

Beginning one day after the catheter insertion, the pregnant ewes were given daily 50

mg of FX (5 ml of 10 mg/ml) via the inserted catheter. FX was injected every day in the

morning around 10:00-11:00 am for 14 days or until the ewes gave birth, whichever came

earlier. Signs of imminent parturition in the ewe (bilateral concave flanks, restlessness,

pawing at the pen floor, moving in circles and looking toward the back) and the lambs’ birth

were observed by DVR from infrared cameras which will be described more detail in the

section DVR and behavioral evaluation. Every attempt was made to measure the lambs’

weight, head circumference, tissue spO2 and to obtain a jugular vein blood sample (via

percutaneous puncture) as soon as they were born. They were dried with a towel, sexed,

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weighed and had on actiwatch around their neck right after birth (more details about

actiwatch in the section Actiwatch/Rest activity monitoring). Lambs were monitored for 14

days and daily weight, tissue spO2, head circumference and abdominal circumference were

measured. They were housed in a smaller pen with their mothers (8x8 feet) to facilitate ewe-

lamb bonding until the end of the monitoring period. On postnatal day 2, 5, 10 and 14, ECG

measurements and blood samplings for FX, NFX concentration were performed (more details

in the sections below).

6.2.3 Digital Video Recording And Behavioral Evaluation:

Two infrared video cameras were installed at two corners of each pen in three pens in

the sheep holding room and connected to a digital video recording system on a computer

which was located on the hallway just outside the sheep pen. Another mobile infrared camera

was used to directly observe and record the lambs’ behavior while they were on the sling for

ECG measurement. The infrared function allowed the recording and monitoring of the ewe

and lamb’s behavior at night without interrupting their circadian rhythm. These cameras

could also be accessed remotely from home and the video records could be analyzed off-line.

Lamb’s activities were monitored continuously at least 14 days to determine any

abnormal behavior changes in the lambs. The number of daily sleep and suckling bouts were

identified from the DVR observations for each lamb from postnatal day 2 until day 10. The

total activity bout was defined as a sum of suckling bout and active bout without suckling

during 24h of daily observation.

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6.2.4 Actiwatch Rest/Activity Monitoring:

A similar method and analysis were used as described in chapter 2. The lambs’

activities were recorded for at least 14 days after birth.

6.2.5 Powerlab Data Acquisition System:

ECG measurement was conducted on the lambs for 1 hour on postnatal day 2, 5, 10

and 14. Lambs were taken to the procedure room for ~ 20 minutes to glue on surface ECG

electrodes. Three ECG snap electrodes were attached to the skin on the back and bilaterally

on the chest and secured in place by adhesive bandages. Lambs were put in the cloth sling,

which located right in front of the holding pen from where the lamb came in full view of the

mother, and the ECG electrodes were connected to a polygraph recorder (model TA-4000,

Gould Instrument Systems Inc, Valley View, OH) and to a Powerlab data acquisition system

(AD instrument, Mountain View, CA, USA) by three snap leads to record their

electrocardiograph. HRV data were analyzed using the same method as described in chapter

2.

6.2.6 Pulse Oximeter: Tissue SpO2 And Heart Rate:

Daily tissue spO2 and heart rate were measured using a pulse oximeter (Nonin

Medical Inc, Plymouth, MN, USA), with the oximeter sensor clipped on the ear of the lamb.

The lamb’s ear was shaved for a better signal. The value is an average of 5 minutes

monitoring.

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6.2.7 FX And NFX Concentration:

Blood samples were also collected after birth from the ewes and lambs and on

postnatal day 2, 5, 10 and 14 from the lambs for fluoxetine measurements. Samples were

taken from the ewes via the inserted jugular vein catheters. Lambs were taken to the

procedure room and the wool along the jugular vein was shaved. The skin was disinfected

with alcohol 70% and Xylocain 2% gel was applied. The jugular vein was accessed by

percutaneous puncture. Approximately 4 ml of blood was collected in Lithium Heparin

(68USP units) vacutainer tubes (BD Vacutainer, NJ, USA) and centrifuged for 20 min at the

speed of 4000rpm in a Centrifuge 5810R (Eppendorf, ON, Canada). Plasma was then

transferred to glass tubes for fluoxetine samples and stored at 0oC until analysis. Lambs

usually slept or lay quiet while blood was collected and were returned to their mother

immediately.

FX and NFX concentrations were measured using a validated liquid chromatography

with tandem mass spectrometry method (LC/MS/MS) (Chow et al., 2011) as described in

Chapter 2. The sample volume used for each enantiomer (R)-, (S)- fluoxetine and (R)-, (S)-

norfluoxetine was 250 µl. The limit of quantitation was 1 ng/ml.

6.2.8 Statistical Analysis:

The areas under the curve (AUC) were calculated for daily variables (weight, sPO2,

heart rate) by applying the formula described by (Altman, 1991). Then the AUC of each

variable were compared between the control and FX group using the unpaired t-test. The

effect of FX exposure and sex on gestational age, birth weight and the time for the lambs to

first stand, walk and suckle were determined using 2 way ANOVA. Two-way ANOVA with

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repeated measures was used to determine the effect of FX exposure and sex on activities

(actiwatch and DVR observations) for the first 10 days of life and on HRV variables on

postnatal day 2,5,10 and 14 in the lambs with multiple comparison correction (Bonferoni t-

test). All data were analyzed using the Sigma Plot 11 statistics software (Systat Software,

Inc; San Jose, CA, USA).

6.3 Results:

In the FX group, ewes were given daily 50mg FX i.v injection for approximately 2

weeks before their due date. The average duration of antenatal FX exposure in the lambs was

12 ± 1.3 days.

Table 6.1 describes the gestational age (GA) and birth weight of the lambs included

in the study in this chapter. There were no significant differences in GA at delivery between

the control and FX-exposed group. However, it was noticed that 2 ewes that delivered

singleton lambs in the FX group had a shorter gestational length (141 and 142 days) as

compared to the normal GA of 147 days at term. In the control group, two lambs from the

triplet also had a shorter gestational length (142 days). There was also no significant

difference in birth weight between control and FX exposed lambs.

Figure 6.1 illustrates daily postnatal weight gain for the first two weeks of life in the

control and FX groups. No significant differences in postnatal weight gain were found

between the two groups.

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Control lamb

GA at birth

(days)

Birth weight (gram)

Lamb number

FX-exposed Lamb

GA at birth

(days)

Birth weight (gram)

Lamb number

L620F 146 5830 Single L756M1 147 4700 Twin L371M 148 6250 Single L752M1 148 4400 Twin L310M1 142 3300 Triplet L752M2 148 5000 Twin L310M3 142 3500 Triplet L577M 141 4100 Single L825M1 145 4900 Twin L630F 142 5000 Single L825M2 145 5300 Twin L756F2 147 3900 Twin L752F 144 5200 Single L643F 148 7000 Single Mean 145 4897 146 4871 SEM 1 421 1.1 388

Table 6.1: Gestational age and birth weight in the control and antenatally FX-exposed lambs M = male, F = female, 1,2,3 after M,F indicates the order of delivery in twin and triplet lambs

FIG. 6.1: (A) Daily weight in the control (n=7) and prenatal FX-exposed lambs (n=7) from postnatal day 1 (day of birth) to postnatal day 14.

Plasma FX, norfluoxetine (NFX) enantiomer concentrations in the ewes (n = 5) at

delivery and in the lambs (n = 7) at birth, on postnatal day 2, 5, 10 and 14 are presented in

Figure 6.2. At delivery, the mean maternal FX and NFX level were 627.9 ± 249 and 66.6 ±

14.4 ng/ml, respectively. Mean plasma FX level was quite low in the lambs at birth, with an

average of 17.3 ± 10.9 ng/ml and decreased progressively thereafter. It became undetectable

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after postnatal day 5. NFX was undetectable in the lambs from birth to postnatal day 14,

which suggests that lambs of this age are unable to metabolize the drug. The relationship

between the lamb: ewe ratio for FX concentration and the duration from the last maternal FX

dose is shown in figure 6.3. The ratio was highest in the lambs that had the shortest duration

from the last FX dose, within 8 hours and it fell rapidly thereafter.

Figure 6.4 illustrates the daily tissue sPO2 and heart rate values obtained from pulse

oximeter from postnatal day 1 to 14 in two control lambs and seven FX-exposed lambs. The

results were similar between the two groups. sPO2 over 14 postnatal days fluctuated within

the normal range 90-100% while heart rate decreased progressively in both control and FX

lambs from an average of 234 ± 17 bpm and 230 ± 19 bpm on postnatal day 2 to 187± 6 bpm

and 200 ± 8 bpm on postnatal day 14, respectively.

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FIG.6.2: FX, (R)-FX, (S)-FX and NFX, (R-)NFX, (S)-NFX concentrations in the prenatally FX-exposed ewes (n= 5) and lambs (n=7). Samples were taken at birth in both ewes and lambs and on postnatal day 2,5,10 and 14 only in lambs. PND = Postnatal day [NFX] levels in the lambs were lower than the limit of quantitation (1ng/ml)

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FIG 6.3: The relationship between the lamb:ewe plasma FX concentration ratio and the time from the last FX dose (hours) in the ewe.

 

 FIG. 6.4: Daily (A) tissue spO2 and (B) heart rate measured by pulse oximeter in the control (n=2) and prenatal FX-exposed lambs (n =7) from postnatal day 1 (day of birth) to postnatal day 14.

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The lambs with prenatal FX exposure were more active than the control lambs, which

was reflected by faster first time activities (stand-walk-suck) and an increase in subsequent

activities obtained from actiwatch and DRV observations. (Figs. 6.5-6.7) It took the FX

lambs (n = 7) only half the amount of time as compared to the control lambs (n = 37) to

achieve their first time activities. While the control lambs started to stand at 32 ± 2 mins

after birth, the FX-exposed lambs did this at 17 ± 3 mins, p = 0.017. In general, the control

lambs attempted to suck within the first one hour of life (56 ± 5 mins) whereas the FX-

exposed lambs did this within the first half hour (29 ± 6 mins), p = 0.006. There was no sex

difference in the first time activities.

Figure 6.6 shows the activity variables from actiwatch monitoring from postnatal day

2 (the first full day after birth) to postnatal day 14 in seven control and seven FX-exposed

lambs. The lambs’ activity increased over days in both control and FX groups, but it was

increasingly greater in the FX group than the control group. Total activity score and active

bout per days were significantly higher in the FX-exposed group from postnatal day 4 to 14

as compared to that on each corresponding day in the control group, while they were

comparable between the 2 groups on postnatal day 2 to 3. Mean score in active periods were

also higher in the FX group vs control group, but not significantly different. In contrast,

episode duration was significantly lower in the FX group compare to the control on postnatal

day 5, 9, 10 and 12. The percentage of time moving was significantly higher in the FX group

compare to control on postnatal day 12. A reverse situation occurred with the percentage of

time immobile.

From DVR observations, sleep, suckling and non-suckling activity bouts were

determined from postnatal day 2 to day 10 in the same seven control and FX-exposed lambs

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and are shown in figure 6.7. The number of total activity bouts is the sum of suckling bouts

and non-suckling activity bouts. Prenatal FX-exposed lambs appeared to have a significantly

lower number of suckling bouts than the control lambs on postnatal day 2, 5 and 6. However,

the AUC of non-suckling activity bouts for 10 postnatal days was significantly higher in the

FX-exposed lambs compared to the control. Therefore, the number of total activity bouts (=

suckling bouts + non-suckling active bouts) was comparable between the two groups until

postnatal day 9 and so was the number of sleep bouts. On postnatal day 10, the number of

total activity bouts was significantly lower in the FX group than the control, and this

difference is due to the significantly lower number of sleep bouts in the FX group than the

control group. There were no significant differences in the number of suckling bouts between

the two groups on postnatal day 10. Collectively, the results suggested that lambs exposed to

FX prenatally were more active than the control group, but the increase in their activity did

not contribute to their feeding behavior and thus, their postnatal weight gain. In addition, we

did not observe any other abnormal behaviors in the FX-exposed lambs for the first 14 days

of life.

Interestingly, the high level of activity appeared to exist in the FX-exposed lambs

even at a low level of FX and undetectable NFX level and this high level of activity still

persisted on postnatal day 10, 14 when the level of FX was no longer detectable. (Figs. 6.2 &

6.6)

HRV was only significantly different on postnatal day 2 and was comparable on

postnatal days 5, 10 and 14 between FX and control groups. The time domain variables of

HRV (SDNN, SD Delta NN, RMSSD) was significantly lower in the FX-exposed lambs than

the control lambs at both 30 and 60 mins of the recording period. Similarly, the total power

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was significantly lower in the FX group but no significant differences were found with LF

power, HF power and LF/HF ratio. (Fig. 6.8) Total activity score from actiwatch during the

1h period of ECG monitoring also showed a lower activity level in the FX group compared to

the control group, however the difference in total activity score between the two groups was

not statistically significant. (Fig. 6.8)

FIG. 6.5: Neonatal behavior (first time to stand, walk, suck immediately after birth) in the control lambs (n = 37) and prenatal FX-exposed lambs (n =7). Asterisk (*) indicates significant differences between the control (open bars) and FX group (black bars), p < 0.05.

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FIG. 6.6: Daily activity variables (active bouts per days, total activity score, mean score in active, episode duration, % time mobile, % time immobile) obtained from the actiwatches in the control (opened, diamond symbols) and prenatal FX-exposed lambs (closed, circle symbols) from postnatal day 2 (the first full day) to postnatal day 14. * indicates significant differences in the corresponding values between the control and FX group, + indicates significant differences from the day 2 within the control group, ^ indicates significant differences from the day 2 within the FX group. Stats: 2 way ANOVA repeated measures with multiple comparison correction Bonferoni t-test

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FIG. 6.7: Daily sleep, suckling and non-suckling activity bouts obtained from DVR observations in the control (open, diamond symbols) and prenatal FX-exposed lambs (closed, circle symbols) from postnatal day 2 (the first full day) to postnatal day 10. * indicates significant differences in the corresponding values between the control and FX group, + indicates significant differences from the day 2 within the control group, ^ indicates significant differences from the day 2 within the FX group. AUC non-suckling activity bout were significantly higher in the FX group (p = 0.04)

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FIG. 6.8: HRV (time domain: SDNN, SD Delta NN, RMSSD; frequency domain: total power, LF power, HF power, LF/HF) on postnatal day 2 in control group (diamond symbol, dotted line, n = 8) and prenatal FX-exposed lambs (circle symbol, solid line, n = 7). * indicates significant differences between the corresponding values in the control and FX groups. + indicates significant differences between the values at 30 and 60 min within the control group, ^ indicates significant differences between the values at 30 and 60 mins within the FX group, p < 0.05. Stats: 2 way ANOVA repeated measures with multiple comparison correction Bonferoni t-test

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    Day 2  Day 5  Day 10  Day 14 

Heart Rate           

Control  Mean  220  4.0  ‐17.8  ‐29.7 

  SEM  4.3  12.5  4.8  2.4 

FX  Mean  225  8.3  ‐22.4  ‐26.3 

  SEM  6.0  0.2  0.6  ‐0.1 

SDNN           

Control  Mean  22.5  ‐2.6  5.9  7.1 

  SEM  2.3  ‐0.2  0.9  2.2 

FX  Mean  15.5* 

7.2  13.1  10.3 

  SEM  0.8  2.9  0.9  1.5 

SD Delta NN           

Control  Mean  18.8  ‐7.5  ‐2.8  ‐4.6 

  SEM  4.8  ‐2.4  ‐1.2  0.2 

FX  Mean  7.2* 

12.0  6.0  2.0 

  SEM  1.4  5.5  1.9  1.3 

RMSSD           

Control  Mean  18.8  ‐7.5  ‐2.8  ‐4.6 

  SEM  4.8  ‐2.4  ‐1.2  0.2 

FX  Mean  7.2*  12.0  6.0  2.0 

  SEM  1.4  5.5  1.9  1.3 

LF Power (ms²)         

Control  Mean  73.8  24.6  96.6  197.2 

  SEM  11.5  14.0  15.3  24.0 

FX  Mean  37.5  52.2  114.3  170.6 

  SEM  4.7  11.7  18.6  24.4 

HF Power (ms²)         

Control  Mean  51.4  ‐25.0  3.0  33.1 

  SEM  13.4  ‐8.8  ‐1.7  11.1 

FX  Mean  19.2  35.0  35.2  25.0 

  SEM  4.8  16.9  8.6  2.6 

LF/HF           

Control  Mean  2.2  1.5  2.0  2.1 

  SEM  0.6  0.1  0.3  0.4 

FX  Mean  2.6  ‐0.2  0.7  2.9 

  SEM  0.5  ‐0.1  0.1  0.5 

           

Control  N =   8  5  12  5 

FX  N =   7  7  7  7  Table 6.2: Mean ± SEM of HRV values for the first 30 mins of the experiment in the lambs on day 2 and the changes from day 5, 10, 14 compared with the values on day 2 (*) indicates significant differences between FX and control group

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6.4 Discussion:

In this study we found that the lambs with prenatal FX exposure were more active

than the control lambs. This was indicated by a faster first time activities (stand-walk-suck)

which represent important neonatal behaviors in sheep and by an increase in subsequent

activities obtained from actiwatch and DRV observations. HRV variables on postnatal day 2

were lower in the FX-exposed lambs than the control lambs. There were no significant

differences in gestational age and birth weight between the two groups. Daily postnatal

weight gain, tissue sPO2 and heart rate obtained from pulse oximeter were also comparable

between the two groups.

Maternal FX level at delivery in our study was higher than that reported in the human

(Kim et al., 2006) and in a previous sheep study in our lab (Morrison, 2001; Morrison et al.,

2002). As was discussed in previous papers (Alfaro et al, 2000; Kim et al., 2004), since

CYP2D6 is a key enzyme involved in FX metabolism, the maternal FX concentration might

be a result of FX-mediated CYP2D6 inhibition. From the literature, the level of FX in the

neonates varies between studies and depends on the postnatal day on which FX was

measured (Kim et al., 2006; Kwon & Lefkowitz, 2008; Rampono et al., 2004; Spencer,

1993). One case report gave a FX concentration at delivery of 26 ng/ml and undetectable

levels at 96h thereafter in an infant, which is similar to our current study (Spencer, 1993).

The FX level in our lambs at birth (mean = 17.3 ± 10.9ng/ml) was lower than that measured

in umbilical cord vein at delivery (mean = 41.3, 95%CI = 16.9, 59.8) from 9 pregnant women

on FX therapy in another study (Kim et al., 2006). Interestingly, NFX was also not detectable

at birth and the days after, which suggests that fetal and newborn lambs are unable to

metabolize the drug. Our result is in agreement with previous study in our lab in which NFX

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was also undetectable in the lamb fetuses after fetal FX administration, in contrast with the

mother (Kim et al., 2004). Since the lamb was not able to metabolize the drug, one would

expect to see a higher level of FX in the lambs than it was. However, we also found that there

was a relationship between the ratio of lamb:ewe FX concentration at delivery and the

duration from maternal the last FX dose (Fig. 6.3), indicating a link between maternal and

fetal FX level. This suggests that following the period of maternal FX administration, the

elimination of FX from the fetus was largely the result of transfer of the drug back to the

mother and maternal elimination.

In the human newborn, motor activity is one of the indicies of biobehavioral

assessment. In sheep, neonatal behavior is one of the many factors contributing to lamb

survival. Lambs that are slow in standing and suckling after birth have a lower survival

chance. (Dwyer et al., 2005) Moreover, first time activities such as standing, walking and

suckling are indicators of neonatal adaptation to postnatal life (Dwyer, 2008b). In the present

study, we found that FX-exposed lambs were faster to stand, walk and suckle as compared to

the control lambs. This suggests that FX-exposed newborn lambs do not appear to have any

difficulty in neonatal adaptation. Our result was different from a human study reported by

(Pedersen et al., 2010) in which the first time to sit and walk was delayed in infants exposed

to antidepressants during second and third trimester of gestation. However, in their study

different classes of antidepressants (SSRIs, TCAs) were included.

For the first 2 weeks of life, there was a progressive increase in the level of lambs’

activity in both the FX-exposed and control groups, which is similar to our developmental

study presented in chapter 3 and the results reported by Rurak et al., (2008). However, FX-

exposed lambs were more active than the control lambs, reflected in significantly higher

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values for both the number of active bouts per day and total activity score between FX and

control groups from postnatal day 4 to 14. Although the FX-exposed lambs were more active,

it appeared that their movement bouts were shorter than the control lambs, which was

reflected by significantly lower values for episode duration in the FX group on postnatal day

5,9,10 and 12. Similar to our findings, there is one study in human infants that also found

prenatally SSRIs exposed infants increase their motor activity during sleep compared to the

control (Zeskind & Stephens, 2004), however the difference in activities between the groups

in their study was attributed to the shorter gestational age in the SSRIs-exposed infants. In

our present study, mean gestational age at delivery was similar between the two groups,

suggesting that the increase in the level of activity in the FX-exposed lambs is independent of

gestational age. Moreover, in our study changes in activity in FX-exposed lambs were not

confounded by additive effects of maternal depressive status or stress during gestation as in

human study. Instead, in sheep ewe and lamb bonding after birth is the key factor affecting

behavior (Dwyer, 2008b) and it appeared that all the ewe-lamb bonding in our sheep was

successfully established soon after delivery. As it was shown in chapter 3, the more vigorous

activity of the lambs for the first month of life was due to their feeding activity. However,

observation from DVR revealed that the increase in the level of activity in FX-exposed lambs

was not due to the increase in suckling activity, which is the major activity in lambs in the

first month of life. Instead, the increase in the level of activity in FX-exposed lambs was due

to the higher in the number of non-suckling activity bouts in the FX group reflected by

significant differences in AUC of non-suckling activity bouts between FX and control

groups. This is consistent with our finding that there was no significant difference in

postnatal weight gain between the two groups. From DVR observation, on postnatal day 10,

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FX-exposed lambs had significantly lower number of sleep bouts as compared to the control,

suggesting they sleep for longer duration than the control. However, on this postnatal day 10,

FX-exposed lambs had a shorter episode duration from actiwatch measurement. This

suggested that even though the FX-exposed lambs slept more, their sleep was more disrupted

than the control. However we did not observe abnormal behavior in the prenatal FX-exposed

lambs during the first 10 postnatal days.

As was mentioned previously, the mechanisms of poor neonatal adaptation in

newborns exposed to SSRIs during gestation are still a matter of debate. From our study it is

apparent that the high level of activity in our FX-exposed lambs was not due to FX toxicity

since the average of FX level in the lambs at delivery was lower than the minimum range of

adult human therapeutic level, which is 40-250 ng/ml (Krogh CME, 1995) and NFX, a

pharmacologically active metabolite, was also undetectable. The question is whether this is a

result of a withdrawal effect. In adults with opioid-withdrawal syndrome, the symptoms can

last for weeks after abrupt discontinuation of the drug involved. Methadone appears to have a

longest duration withdrawal symptoms among opioids after acute discontinuation with the

severity of symptoms and can last up to 16 days after the last dose. (Kosten & O'Connor,

2003) The argument is that in my study, the increased activity in the lambs in the FX groups

was observed up to 2 weeks after birth, which can be similar the duration of opioid-

withdrawal syndrome in the human. Longer follow-up of the increased activity in the

antenatally FX-exposed lambs will help to rule out the mechanism. That is, if the increased

activity in the lambs still persisted after 2 weeks, it is less likely due to the withdrawal

syndrome.

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However, I believe that the increased in activity in the FX-exposed lambs is less

likely due to withdrawal syndrome. Rather, it is due to a change in the fetal brain

development since evidence from human and sheep studies have shown that the alteration in

activity resulting from antenatal SSRI exposure seems to appear in the fetuses before the

drug exposure ended and continue until the postnatal period. Mulder et al., (2011) reported

an abnormal phenomenon of continual bodily activity during non-REM sleep in SSRIs-

exposed fetuses. Similarly, another study found SSRIs-exposed fetuses at 26 weeks of

gestation had more jerky movements than the control and MDD/no SSRIs exposure groups

(Salisbury et al., 2006). In sheep, there were also increased eye movement and fetal breathing

movement with 8 day FX i.v infusion. (Morrison et al, 2001) Collectively, these findings

from our study and others support the hypothesis that a possible underlying mechanism for

the high level of activity in the FX-exposed lambs is SSRIs-induced changes in fetal brain

development. Further evidence for this mechanism comes from studies showing a lower cord

blood level of calcium-binding protein S100B, a biomarker of early brain development and

central serotonergic function, in SSRI-exposed neonates (Pawluski et al., 2009). It is known

that the astroglial 5-HT 1A receptor is activated by an increase of synaptic 5-HT after SSRI

administration, and this will stimulate a release of S100B protein, which in turn mediates the

growth and survival of neurons (Whitaker-Azmitia et al., 1990; Whitaker-Azmitia, 2001).

In our study we also found that the time domain of HRV (SDNN, SD Delta NN,

RMSSD) and total power were significantly lower in the prenatally FX-exposed lambs on

postnatal day 2 compared to the control lambs. It is known that changes in HRV are usually

associated with physical activity (Nakamura et al, 1993; Voss et al, 2002; Warren et al,

1997). On postnatal day 2, the 24 h total activity score were comparable between control and

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FX groups. Further analysis of the total activity score for during the 1h of HRV measurement

on postnatal day 2 did not show significant differences in activity score between the two

groups even though the activity score during this 1h of HRV measurement was lower in the

FX-exposed group (Fig. 6.8). Thus, it is uncertain whether activity plays a role in the lower

level of HRV in the FX-exposed group. There are limited data on HRV changes in the

newborns after prenatal SSRIs exposure in the literature, although two studies did report a

similar trend of lower HRV rhythms in the newborns or a blunted facial and HRV response

to pain in 2 month-old infants after prenatal SSRI exposure (Oberlander et al., 2005; Zeskind

& Stephens, 2004). Since changes in HRV in our study only occurred in the time domain but

not in frequency domain indices, it is uncertain if the changes come from sympathetic or

parasympathetic systems. Changes in HRV also occurred in SSRI-exposed human fetuses. A

recent study has reported that short and long-term variation of fetal heart rate, accelerations

and duration of high variability episodes did not change during the day and were lower in the

SSRIs-exposed fetuses compared to a significant increase during the day in the control group.

(Rurak et al., 2011) Taken together, our results and the other findings showed a similar low

HRV or blunted HRV response in the SSRIs-exposed infants which originate from the fetal

period with SSRI exposure.

The major effects of prenatal FX exposure on the postnatal lambs in the present study

consisting of an increase in activity at birth and from postnatal day 4 to 14 and low HRV on

postnatal day 2. There was a lack of FX effects on gestational length, birth weight, postnatal

weight gain. Heart rate and tissue SpO2 were also not different between the control and FX

groups. Our results are different from other previous human studies which reported an

increase in preterm delivery, shorter gestational length, lower birth weight and postnatal

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weight gain in third trimester FX exposure fetuses. (Chambers et al., 1996; Goldstein, 1995;

Hendrick et al., 2003; Lewis et al., 2010; Oberlander et al., 2006; Suri et al., 2007). However,

our results were consistent with those found in previous sheep study in our lab (Morrison,

2001; Morrison et al., 2002) except for postnatal weight gain which was not obtained in

Morrison el al study. As was discussed before, different results between human studies and

ours are probably due to the presence of confounding factors in human studies but not in

animal studies, for example the maternal depressive illness and other associated risk factor

such as smoking and alcohol abuse. Even though maternal illness severity was accounted for

in the Oberlander et al and Suri et al studies, it is possible that the cumulative effect of FX

exposure over most or all of pregnancy in the human studies, compared to the short-term late

gestation sheep studies, resulted in the observed lower birth weight and gestation length.

In summary, from this study we found that the newborn lambs were not able to

metabolize FX, thus FX metabolise and clearance in the fetal lambs were likely achieved via

the mother. FX-exposed lambs were more active than the control lambs at birth and from

postnatal day 4 to 14 and had a lower HRV on postnatal day 2. Together with the FX

measurement after birth and on postnatal day 2, 5, 10, 14, the increase in activity in the

postnatal lambs exposed to the drug in utero suggests the most likely mechanism for these

effects is alteration in fetal brain development, due to the importance of serotonin in brain

maturation. However, DVR observations showed the increase in activity score was not due to

suckling activity but due to a decrease and disrupted sleep activity. Consistently, there was

no difference in postnatal weight gain in the two groups. And there was also a lack of FX

effect in gestational age, birth weight, heart rate and tissue SpO2.

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7. Summary and Conclusions

Depression has been recognized as one of the most burdensome disorders during

pregnancy, affecting at least 10-15% of pregnant women (Bhatia & Bhatia, 1999; Nonacs &

Cohen, 2003). Pharmacological treatment is a norm. And one third of pregnant women

suffering from depression take SSRIs as antidepressants (Oberlander et al., 2006). There is an

increasing use of FX and other SSRIs during pregnancy (Andrade et al., 2008). A

considerable amount of attention has been paid in the literature to the assessment of birth

outcomes following in utero FX exposure during late gestation. However, studies that focus

on the mechanisms underlying the observed outcomes are relatively sparse. Several

mechanisms have been proposed but there is a lack of supporting evidence. Previous

experiments in our lab were conducted with 8 day maternal FX i.v infusion to expose late

gestation sheep fetuses to FX and primarily focused on the fetal responses and the underlying

mechanisms. (Morrison et al., 2001; Morrison, 2001; Morrison et al., 2002; Morrison et al.,

2004; Morrison et al., 2005). The current study is in part a continuation of the previous

studies, focusing on the effects of maternal FX treatment on postnatal lambs and the

underlying mechanisms of poor neonatal adaptation. As noted elsewhere in this thesis, this

syndrome occurs in 30% of newborns prenatally exposed to SSRIs (Koren et al., 2009). In an

attempt to elucidate the underlying mechanisms for poor neonatal adaptation, I set out to

determine if SSRI toxicity is the responsible mechanism, FX was given to the newborn lambs

acutely on approximately postnatal day 4 in an attempt to reach the plasma concentration

levels observed in human infants at the same age. This was successful in that the level of FX

in the newborn lambs fell within the range of plasma FX concentration measured in human

infants sampled on postnatal day 2 following in utero exposure to the drug. However, our

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protocol of acute, single dose FX administration differs from the clinical setting since human

infants are exposed to FX throughout all or most of pregnancy. Similarly, when FX is

prescribed to treat depression in children and adolescents, it is given chronically with

multiple, daily doses. However, in my subsequent study to investigate the effects of maternal

FX treatment on the postnatal lambs, FX was given to the pregnant ewes for the last ~2

weeks of pregnancy, but again for a relatively short period of gestation. The lambs were

exposed in utero to FX at clinically relevant concentrations, followed by evaluation of birth

weight, postnatal weight gain, behavioral development and cardiovascular function. In the

previous studies in our lab, FX was administered to the ewes using infusion pump and thus

required surgical instrumentation of the ewes and fetuses, and the restraint of the ewes in a

monitoring pen during the infusion and monitoring period (Morrison et al., 2001; Morrison et

al., 2002; Morrison et al., 2004; Morrison et al., 2005). Thanks to the procedure to implant

jugular vein catheter subcutaneously in the pregnant ewes, we were able to successfully

administer FX allowing the ewes to moving freely in their pen. FX was given i.v to the

pregnant ewes once daily to mimic the human dosing with pulsatile FX treatment. Oral

administration of the drug, which is the norm in humans, was not attempted in this and

previous studies because of the concern about the impact of ruminant nutrition on absorption

of orally administered drugs in sheep.

The following conclusions can be made from the results of the current study:

1. The results from the developmental study showed a decrease in heart rate,

increase in arterial pressure and maturation of the Autonomic Nervous System (ANS)

system as the lambs aged. pH, pO2, O2 saturation increase, while pCO2, glucose, lactate,

Hb, urine flow rate decrease with age. The plasma cortisol level is high around the time

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of birth and decreases progressively thereafter. The 1h activity rhythm, which only exists

and is dominant over the first month of life is associated with suckling. The changes in

some of the physiological variables including heart rate, SDNN, glucose, pO2, urine flow

rate occurred around the window of weaning time. Weaning was defined in our study as

the time window between when lambs started to first consume solid food to when the 1h

rhythm due to suckling disappeared. Alternatively, the weaning time could be determined

by measuring the level of plasma ketone bodies (acetoacetic acid and 3-hydroxybutyric

acid) in the lambs. Even though the neonatal rumen epithelium is not ketogenic, the

ruminal epithelium in a mature ruminant animal is able to produce ketone bodies, which

is supplied by the microbial fermentation of feed (Lane et al., 2002). In addition, the

developmental data also showed that at 3 days of age male lambs are slightly more

acidotic than female lambs. This is similar to the findings of Ishak et al in lambs

delivered preterm at 133 d, although in this study male lambs exhibited other differences

from female lambs, including a higher pCO2, blood lactate and glucose concentrations

and higher arterial pressure (Ishak et al., 2012). In preterm lambs, this reduced postnatal

cardio-respiratory adaptation in male lambs could contribute to a higher mortality rate in

male than female neonates, especially related to respiratory distress syndrome

(Ingemarsson et al., 1997; Ingemarsson, 2003; Khoury et al., 1985). The current data

suggest even with delivery at term, respiratory function or acid-base regulatory

mechanisms in male lambs are less effective than in females, although the sex differences

are much less than with preterm birth. At 3 days of age, LF power were also higher in

male than female lambs, possibly due to an influence of testosterone, which present in the

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male lambs by 36h after birth (Savoie et al., 1981), on the ANS or due to an ANS

response to an unfavorable environment (i.e. lower pH level).

However, one limitation of the developmental study is that the variables were not

measured on a daily basis, and thus cannot provide an exact time when the changes occur

as well as the pattern of changes between the experimental intervals. In addition, the

lambs included in the developmental study were also used for a longitudinal study of FX

effects, although only as control animals.

2. Acute FX administration did not cause any significant effects on the feeding

behavior, rest-activity cycles, cardiovascular and respiratory functions and the cortisol

level in the ~ 4 day old postnatal lambs. Even though arterial blood pressure increased for

the first 10 mins and heart rate decreased at 2 mins after FX injection, the changes were

too minor and transient to have any clinical or physiological significance. Therefore, it is

less likely that SSRIs toxicity is the mechanism underlying poor neonatal adaptation in

human infants exposed to this drug and other SSRIs in utero. This is further confirmed by

the subsequent results.

3. Acute FX administration did not cause any significant effects in the lambs at

10 days and 3 months of age. However, at 1 month of age, transient hypoxemia occurred

at 5 mins after FX injection. The hypoxemia was more severe in males than females.

Lambs were also less active on the day of FX administration at this age. Similarly, at 6

months of age, transient hypoxemia also occurred at 5 mins after FX injection but there

was no sex difference. Behavioral observations showed lambs were less active but more

irritable following FX administration. At 1 year of age, arterial pressure increased

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significantly for 30 mins following FX injection. The increase was more profound and

lasted longer in males. HRV increased at 30 mins after FX and hypoxemia also occurred

at 5 mins after FX injection. However, no changes in the HPA axis function were

observed. FX effects on arterial pressure and blood oxygenation only occurred in the

older lambs but not in the younger lambs potentially due to the lower level of platelets,

which are the main reservoir of peripheral 5-HT in the younger lambs. In humans, it has

been known that the platelet concentrations are lower in newborns and children than in

adults (Merritt & Davidson, 1933) and platelet 5-HT concentration in newborn is only ~

42% of the value in children and adults (Flachaire et al., 1990). Measurement of platelet

concentration, platelet 5-HT concentration and the magnitude of transient increase in

blood 5-HT concentration following FX administration in sheep as a function of age

could confirm this hypothesis. However, analysis of serotonin from plasma samples is

technically difficult. In this regard, I did attempt to measure plasma 5-HT concentration

after FX injection in several lambs at 3 months of age but the results were very variable.

This was likely due to platelet/aggregation during sample collection, resulting in release

of serotonin from the platelets into blood. The sex differences in FX effects on arterial

pressure in the older lambs could be due to the effect of ovarian steroids on increasing 5-

HT clearance by the lung in the females, leading to a lesser transient increase in blood 5-

HT levels following FX injection (Bakhle & Ben-Harari, 1979; Gillis & Pitt, 1982). In

addition, our data showed that there was a relationship between FX concentration and the

changes in respiratory effects in the 1 year old lambs. The higher the FX concentration,

the greater decrease in O2 saturation. Dose-related effects of SSRIs have also been

observed in human and animal studies. In particular, increased risk of preterm birth was

199

mainly found in women treated with high dose SSRIs during pregnancy (Roca et al.,

2011). Increased fetal behaviors were also observed at standard and high SSRIs doses,

but not at the low dose and the effect at higher doses was greater than at standard doses.

(Mulder et al., 2011) In a rat study, respiratory rate reduced at standard doses and further

decreased at the higher doses of FX, while no changes occurred at the low dose of FX.

(Annerbrink et al., 2010) Moreover, in our study cardiovascular and respiratory effects of

FX were also significantly related. The greater the increased arterial pressure, the greater

the decreased pO2 concentration. As the mechanism of action of FX in inhibiting

serotonin reuptake by platelets leads to an acute increase in plasma serotonin, and since

serotonin is a potent vasoconstrictor, it can cause a sudden increase in blood pressure.

This could result in a decrease in cardiac output due to an afterload effect, which would

reduce pulmonary blood flow and result in the decrease in arterial pO2.

4. Postnatal lambs who were prenatally exposed to FX showed an increase in

activity for the first 2 weeks of life, and a lower HRV (time domain indexes: SDNN, SD

Delta NN, RMSSS) on postnatal day 2. Measures of birth and postnatal outcomes such as

gestational length, birth weight, postnatal weight gain, tissue sPo2 were not affected by

late gestation FX exposure. The increase in activity in the FX-exposed lambs existed in

the absence of increase in suckling activity, which is consistent with no difference in

postnatal weight gain between FX-exposed and control lambs. Measurement of a low

level of plasma FX concentration at birth and on the following postnatal day 2,5,10 and

14 confirmed that FX toxicity is less likely to be the underlying mechanism for poor

neonatal adaptation observed in human infants. Since the significant increase in activity

in the FX-exposed lambs existed until postnatal day 14, while FX level was low from

200

postnatal day 2, a withdrawal syndrome is also less likely to be the underlying

mechanism. Thus this leaves the mechanism of an SSRI-induced alteration in fetal brain

development to be related to the importance of serotonin in brain maturation pathways as

the most likely candidate. Indeed, an increase in fetal activity and a lower HRV through

the day have been observed in the human fetuses exposed to SSRIs in utero (Mulder et

al., 2011; Rurak et al., 2011). Measurement of serotonin concentrations in the

cerebrospinal fluid would provide direct evidence for the effects of FX on the lambs’

serotonin brain levels. Alternatively, S100B protein has been used as a biomarker for

early brain development and central serotonergic function (Pawluski et al., 2009). An

increase in synaptic 5-HT after SSRIs administration activates the astroglial 5-HT1A

receptor, which leads to a release of S100B protein which mediates the growth and

survival of neurons (Whitaker-Azmitia et al., 1990; Whitaker-Azmitia, 2001). Therefore,

in the absence of cerebrospinal fluid samples, determination of S100B protein could

provide a marker for changes in extracellular serotonin caused by FX administration. The

possibility of measuring plasma S100B concentration in my study arose late in the study,

after completion of the developmental studies. Unfortunately, due to the lack of

remaining blood samples in the control group, S100B protein analysis was not performed

in our studies.

In addition, from our studies it appeared that acute postnatal and long-term

prenatal FX exposure exert opposite effects. While acute FX injection caused a reduction

in activity, subchronic prenatal FX exposure resulted in hyperactivity in the lambs. Since

clinically it usually takes about 2 to 4 weeks for an SSRIs fully work, acute FX

administration could elicit a paradoxical antidepressant effect (i.e. sedation rather than

201

hyperactivity), which is different from the long-term exposure. There was a case report of

a paradoxical sedative effects at the onset of FX treatment, but this effect disappeared

after a few weeks of continuous FX therapy (Gupta et al., 1994). Moreover, it has been

documented that FX treatment is associated with a significant increase in the rate of both

activating adverse events including insomnia, agitation, anxiety, nervousness, and

sedating events such as somnolence or asthenia, as compared to placebo treatment in

patients with different baseline psychomotor activity status (agitated, retarded or neither).

Interestingly, 10% of patients that experienced adverse events actually reported both

effects, either simultaneously or serially. (Beasley et al., 1991)

Alternatively, it has been shown in a previous study in our lab that an enhanced

prepartum cortisol surge is associated with long-term prenatal FX-exposure in the fetal

lambs (Morrison et al., 2004). This could result in an early maturation of the musculo-

skeletal system, and thus explain the observed hyperactivity in the postnatal lambs

exposed to FX prenatally in our current study.

In summary, my study is the first to investigate postnatal outcomes in lambs exposed

to FX in utero or postnatally. In this study, we have two study cohorts: one was exposed to

FX acutely after birth and the other was exposed prenatally to FX for 2 weeks of late

gestation, which is equivalent to the last month of third trimester in human pregnancy. Acute

FX administration on postnatal day 4 did not cause any significant effects other than slight

and transient changes in heart rate and arterial pressure. In contrast, in the ~ 2 weeks

gestational FX exposure lambs, we observed a decreased time required for initial lamb

milestones (i.e. first time to stand, to walk and to suckle), a reduced HRV on postnatal day 2,

a hyperactivity not associated with suckling. (see Table 7.1; A vs C)

202

Human studies (Table 7.2 B & C) have shown that antenatal SSRIs exposure results

in many adverse birth outcomes in the offspring such as shorter gestational age, increased

risk of preterm birth, lower birth weight, small for gestational age, reduced postnatal weight

gain, poor neonatal adaptation (respiratory difficulty, cyanosis on feeding, irritability), lower

APGAR score, increased admission rate to NICU and reduced endocrine and facial response

to pain stimulus (via vaccination) at 1 and 3 months of age (Chambers et al., 1996; Costei et

al., 2002; Goldstein et al., 1995; Kallen, 2004; Lattimore et al., 2005; Levinson-Castiel et al.,

2006; Lewis et al., 2010; Lund et al., 2009; Maschi et al., 2008; Oberlander et al., 2005;

Oberlander et al., 2006; Oberlander et al., 2008; Roca et al., 2011; Simon et al., 2002; Toh et

al., 2009; Wen et al., 2006; Wisner et al., 2009; Zeskind & Stephens, 2004). However, we

did not observe any differences in the corresponding outcomes reported in human studies

such as gestational age, birth weight, postnatal weight gain in the lambs whose mothers were

administered FX i.v for ~ 12 days late gestation (see Table 7.1 C vs Table 7.2 B).

Differences observed in my study and human studies could be due to several reasons.

In human studies, the infants were exposed to the drugs for all or most of pregnancy, whereas

in my study, the exposure period was limited for ~ 12 days late gestation. In addition, women

who were treated with antidepressants were also clinically depressed; depression per se, and

its associated risk factors such as smoking, alcohol and drug abuse could be major

confounders for adverse effects in the offspring. As far as we know, our sheep were not

depressed. In the current study, we only investigate one subtype of SSRIs, fluoxetine, while

in human studies, many subtype of SSRIs (paroxetine, citalopram, sertraline, fluvoxamine,

etc.) and other antidepressants (TCA, SNRIs) were included.

203

Similar to our results, in one rodent study, there were no differences in body weight at

postnatal day 1, 8, 15, 22 between pups exposed to saline or FX during pregnancy and

lactation (from GA 0 to PND 21) (Lisboa et al., 2007). Other studies in rodents have also

shown changes in postnatal behavior (long-term effects) in mice/rats exposed to SSRIs

postnatally over the range from postnatal day 0 to 21 (see Table 7.3B) (Ansorge et al., 2004;

Hansen et al., 1997; Lee, 2009; Lisboa et al.,2007; Maciag et al., 2006; Manhaes de Castro et

al., 2001; Mirmiran et al., 1981; Popa et al., 2008). For instance, Lisboa et al, 2007 reported

that female FX-exposed mice had an increased immobility in forced swimming tests on PND

30 and 70 while male FX-exposed mice decreased their impulsivity on PND 40 and 70

(Lisboa et al., 2007). In the rat, neonatal exposure to citalopram (SSRI) from postnatal day 8

to 21 resulted in a decreased synthesis of tryptophan hydroxylase in the dorsal raphe and 5-

HT transporter expression in the cortex, increased locomotor activity and decreased sexual

behavior (male) in adult rats. (Maciag et al., 2006). The observed hyperactivity in our

prenatally FX-exposed lambs is similar to the increased locomotor activity in the Maciag’

study but different from the remaining rodent studies which reported an increased immobility

following SSRI exposure. (see Table 7.3 B). Despite this, the results of chronic FX or SSRI

exposure in my study, human studies (Table 7.2 B &C) and rodent studies (Table 7.3 A &B)

showed that there are long-term effects in the offspring exposed to SSRI (FX). These long-

term effects following FX exposure has been identified to be mediated by 5-HT transporter

blockade in rodent models that were exposed to FX early ex utero (PND 4-21) (Ansorge et

al, 2004). Interestingly, the effect of antenatal SSRIs exposure in human and sheep studies

actually appears to emerge in the fetal period before the treatment was ended. Indeed, a

blunted fetal heart rate response during the day, a reduced MCA pulsatile index, increased

204

activities in human fetuses on non-REM sleep, increased jerky movement and lower

reactivity to the vibroacoustic stimulus were observed (Mulder et al., 2011; Rurak et al.,

2011; Salisbury et al., 2006) (see Table 7.2A). In the sheep fetuses, 8 day continuous

maternal i.v FX infusion led to a reduced REM sleep, increase quiet sleep, enhanced

prepartum ACTH and cortisol surge and a transient decrease in uterine artery blood flow

(Morrison et al., 2001; Morrison, 2001; Morrison et al., 2002; Morrison et al., 2004;

Morrison et al., 2005). Taken together, the results from this study contribute to elucidating

the mechanisms of underlying poor neonatal adaptation in humans exposed to SSRI in utero.

So far, data from the current study and other relevant studies have pointed to the mechanism

of SSRIs-elicited altered fetal brain development. Particularly, prenatal SSRIs exposure

might alter 5-HT signaling in utero. This is important in the context of a “fetal programming”

impact on adult health. We hope the data gained from the present study contributes valuable

knowledge, and could assist physicians and pregnant women in the assessment of the safety

of the drugs, and to make the best informed decisions based on the risks and benefits of

antidepressant treatment during pregnancy.

In this study, the postnatal follow-up of prenatal FX exposure was limited only to 2

weeks following birth. Considering the increased activity in the newborn lambs is equivalent

to jitteriness, one of the symptoms of poor neonatal adaptation in human exposed in utero to

the drug, it would be very interesting to follow up their behaviors at longer time beyond this

2 week period, and at milestones of development, such as weaning (at 2 months), puberty (at

6 months) and adulthood (at 12 months) by actiwatch and DVR observations. A study in

human has reported that increased aggressiveness scores in the 4-year old children exposed

to SSRIs during gestation is associated with a history of poor neonatal adaptaion, however,

205

current maternal mood and parental stress appear to be the best predictors for the

externalizing behaviors than a history of prenatal depression or SSRIs exposure. (Oberlander

et al, 2007) The challenge to distinguish between the effects of the drug exposure and a

negative influence of postnatal social environment with depressed mothers on neurocognitive

development in the children in human study might be resolved using our sheep model.

Further studies to confirm the hypothesis of in utero altered 5-HT signaling in

prenatal SSRI exposure could be accomplished by investigating the fetal brain development

in SSRIs exposure subjects. It would be ideal to be able to assess fetal brain structure

development by invasive methods such as Magnetic Resonance Imaging (MRI), Positron

Emission Tomography (PET) and follow-ups evaluating the structure of brain development

and function after birth and up to adulthood. Alternatively, the effect of SSRIs on fetal brain

development could be evaluated by serial measuring plasma S100B protein, a biomarker of

early brain development and central serotonergic function, in the sheep fetuses exposed to

SSRIs (FX) following up to postnatal period into adulthood. In addition, it would be

interesting to investigate the transgenerational effects on fetal programming of altered 5-HT

signaling pathways in utero. Given the time required for a gestation of 145 days and

additional 1-2 years for the female lambs to be able to reproduce, the sheep model is

probably not an appropriate model for these long-term studies. Rodents or guinea pigs might

be better models for this type of study.

206

Table 7.1: Outcomes of chronic antenatal and acute postnatal FX exposure in fetal and postnatal lambs

Sheep studies Agents Outcomes

(A) Present study Acute FX i.v (1mg/kg) on PN

day 4

No differences in feeding behavior, postnatal weight gain, no respiratory difficulties, no

differences in cortisol level

Slight and transient heart rate and arterial pressure changes

(A) Present study Acute FX i.v (1mg/kg) at ~ 10

days, 1,3,6 and 12 months of age

Increased arterial pressure in the 1 year old lambs

Transient hypoxemia in the older lambs (1,6 and 12 months of age)

Decrease activities (1 & 6 months of age)

No changes in HPA axis function

(B) Morrison et al, 2001

(B) Morrison et al, 2002

(B) Morrison et al, 2004

Maternal FX i.v infusion x 8

days (~125-140 GA)

(98.5µg/kg.d)

Altered fetal behavioral state: Increased quiet sleep (low-voltage ECoG activity), eye movements

and fetal breathing movements. Decreased REM sleep (high-voltage ECoG activity)

Transient decrease in uterine artery blood flow

Transient fetal hypoxemia and acidosis for the first 24h of infusion

Increased prepartum ACTH & cortisol surges

(C) Present study Maternal daily FX (50mg) i.v x

~12 days (~131-143 GA)

No differences in gestational age, birth weight

Decreased time required for initial lamb milestones (to stand, to walk, to suckle)

Reduced HRV on PN day 2

PN 2- 14: hyperactivity not associated with suckling, no differences in postnatal weight gain, no

respiratory difficulties

207

Table 7.2: Outcomes of antenatal SSRI exposure in human (exposure for most or all of pregnancy)

(A) Human studies Agents Outcomes

Mulder et al, 2011

Prospective study

SSRIs throughout gestation Fetuses:

Disrupted non-REM sleep

Increase bodily activity during non-REM sleep

Rurak et al, 2011

Longitudinal observational study

SSRIs throughout gestation Fetuses (36 weeks gestation)

Blunted fetal HRV

Lower MCA pulsatility index: MCA mean flow velocity, MCA volume flow, MCA cross-sectional area

Salisbury et al, 2006

Prospective study

SSRIs + no MDD vs MDD

+ no SSRIs vs MDD +

SSRIs vs control

At 26 weeks GA:

Less movement, more jerky movements, lower reactivity to vibroacoustic stimulus

At 36 weeks GA:

SSRI or MDD fetuses: increased reactivity to vibroacoustic stimulus

SSRI + MDD fetuses: decreased reactivity to vibroacoustic stimulus

Newborns:

Increased tremor, startles, hypotonia and lowest quality of movement scores

208

(B) Human studies Agents Outcomes (short-term effects)

Chambers et al, 1996

Prospective/ retrospective reports

FX

Early vs late gestation

exposure

Newborn:

Increase risk of poor neonatal adaptation

Increase risk of special- nursery admission

Simon et al, 2002

Retrospective records

SSRIs

TCA

Shorter gestational age in SSRIs exposed infants

Lower birth weight

Lower Apgar scores (3rd trimester SSRI exposure)

Kallen et al, 2004

Prospective cohort study

SSRIs (Citalopram, FX, PX,

Sertraline)

Increased risk of respiratory distress

Increased risk of low Apgar score

Increased risk of convulsions

Wen et al, 2006

Retrospective cohort study

SSRIs Increased risk of low birth weight, preterm birth, fetal death, and seizures

Wisner et al, 2009

Prospective observational

SSRIs Increased risk of preterm birth in continuously SSRIs exposure across gestation

209

(B) Human studies Agents Outcomes (short-term effects)

Oberlander et al, 2006

Retrospective record-linkage

study

SSRIs (1st trimester to

delivery)

Increased risk of low birth weight

Increased risk of respiratory distress

Roca et al. 2011

Case-control study

SSRIs Increased risk of preterm birth (mainly in high dose)

Lund et al, 2009

Prospective cohort study

SSRIs during pregnancy Increased risk of preterm birth, lower 5-min Apgar score and NICU admission

Lewis et al, 2010

Prospective case-control study

Antidepressants Increased rate of preterm birth, lower birth weight, smaller head circumference

Toh et al. 2009

Retrospective cohort study

SSRIs vs non-SSRIs

antidepressants

Increased rate of smaller than gestational age in late gestational SSRIs exposure

Increased rate of preterm birth and smaller than gestational age in non-SSRIs antidepressant exposure

Levinson-Castiel et al, 2006

Cohort study

SSRIs (PX, FX, Citalopram,

Sertraline, Venlafaxine)

Neonatal abstinence syndrome in 30% SSRIs exposed neonates (Finnegan score)

210

(B) Human studies Agents Outcomes (short-term effects)

Goldstein et al, 1995

Prospective & retrospective

study

FX (3rd trimester) Incidence of poor neonatal adaptation to FX exposure: 13.4%

Increased risk of respiratory signs

Increased risk of sleep problems

Maschi et al, 2008

Prospective cohort study

PX, FX, other

antidepressants

Increased incidence of poor neonatal adaptation but not statistical significant

Costei et al, 2002

Prospective study

Paroxetine (early vs late

gestational vs no exposure

Increased risk of neonatal complications (respiratory distress, hypoglycemia, bradycardia, suckling

problems, prolonged hospitalization)

Increased risk of respiratory difficulties

Zeskind et al. 2004

Prospective study

SSRIs (citalopram, FX, PX,

sertraline)

During pregnancy

Shorter gestational age

Increase motor activity (*) and tremulous

Fewer rhythms in HRV (*)

Fewer changes in behavioral state, fewer different behavioral states and lower peak behavioral state

Increased REM sleep

(*) no longer significant after adjusted for gestational age

211

(C) Human studies Agents Outcomes (long-term effects)

Lewis et al, 2010

Prospective case-control study

Antidepressants At 1 month, lower body weight in exposed infants

Oberlander et al, 2005

Prospective study

SSRIs (FX, PX, sertraline)

(prenatal and postnatal

exposure)

At 2 months of age:

Blunted facial response to heel lance in prenatal SSRI exposure infants

Reduced parasympathetic withdrawal

Increased parasympathetic cardiac modulation during recovery in pre and postnatal SSRI exposure

infants

Oberlander et al, 2008

Prospective study

SSRIs Reduced early evening basal cortisol level

At 3 months of age:

Higher post-stress cortisol levels in SSRIs exposed infants.

212

Table 7.3: Postnatal outcomes of antenatal and postnatal SSRI exposure in rodents

(A) Rodent studies Agents Outcomes

Forcelli et al, 2008 FX

Prenatal infusion for 14 days

PN 30: decreased conflict-exploratory behavior

PN 60: increased place preference

PN 90: increased extinction response rate

PN 120: decreased nucleus accubens cell count & serotonin transporter-like in raphe nucleus

(B) Rodent studies Agents Outcomes

Lisboa et al, 2007 FX

GA 0-PN 21 to dams

7.5mg/kg gavage

No differences in body weight @ PN day 1,8,15,22

PN 30, 70: Increased immobility (female)

PN 40, 70: decreased impulsivity (male)

Lee et al, 2009 FX

PN 0-6

10mg/kg s.c

PN day 7-35:

Neuronal structure deformation in the somatosensory cortex

PN day 30-35

Higher thermal threshold (hotplate test)

Impaired whisker-specific tactile function (gap-crossing test)

Altered exploratory behavior (open field test)

213

(B) Rodent studies Agents Outcomes

Ansorge et al, 2004 FX

PN 4-21

10mg/kg i.p

At 12 weeks of age:

Decreased exploratory behavior in open field and elevated plus maze test

Prolonged latency to begin feeding in novelty-suppressed feeding test

Prolonged latency to escape a foot-shock in shock-escape paradigm

Mashaes de Castro et al,

2001

Citalopram

PN 1-19

20mg/kg s.c every 3 days

PN day 90-120:

Decreased aggressive behavior in footshock test

Hansen et al, 1997 SSRIs (LU)

PN 8-21

2.5-15mg/kg bid

At 4 months:

No difference in open field test & social interaction test

Increased immobile time in forced swim test

Maciag et al, 2006 Citalopram

PN 8-21

5mg/kg x 2 s.c

Adult:

Decreased synthesis of tryptophan hydroxylase in dorsal raphe & 5-HTT expression in cortex

Increased locomotor activity

Decreased sexual behavior

214

(B) Rodent studies Agents Outcomes

Popa et al, 2008 Escitalopram

PN 5

10mg/kg s.c x 2 weeks daily

Sleep anomalies

Anhedonia

Increased helplessness

Increased response to acute stress

Increased serotonergic autoinhibitory feedback

Mirmiran et al, 1981 Chlorimipramine

PN 8-21

x 2 daily

Decreased active sleep, increased quiet sleep

Decreased exploratory behavior in open field test

Decreased temporal learning task

Increased spatial task

215

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Appendices

Appendix 1: Actiwatch Variables Data In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age:

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. ~4 day 10 day 1M 3M 6M 1Y

N = 6-11 Mean

SEM

N = 9-10 Mean

SEM

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 4 Mean

SEM

N = 5-6 Mean

SEM

Active bout per day Normal day in pen 63.3 4.24 47.6 3.93 44.6 3.62 36.0 4.81 33.7 3.40 Average of 3 or 4 days 57.3 5.14 45.4 2.97 48.7 3.19 41.4 4.76 34.5 3.07 Control exp 40.2 3.49 61.3 5.84 48.1 4.28 33.7 2.81 24.9 4.88 20.8 2.64 FX exp 42.4 4.78 55.3 5.08 35.8 2.81 33.4 4.50 28.0 5.05 18.8 1.76 Post FX d1 47.5 3.22 60.2 5.89 45.7 3.57 37.8 4.80 31.1 5.39 23.8 2.67 Post FX day 2 49.7 3.97 58.5 4.60 45.0 3.45 35.4 3.85 24.8 4.16 20.0 2.09 Post FX day3 48.0 4.47 58.8 4.97 43.6 3.00 43.5 3.15 42.2 4.23 42.1 6.38 Mean score in active Normal day in pen 126.6 5.76 84.5 5.82 62.5 4.70 58.4 3.76 55.8 7.00 Average of 3 or 4 days 116.5 8.39 85.0 4.61 66.7 3.37 63.2 4.27 56.7 6.47 Control exp 95.2 8.51 118.7 8.84 84.0 6.85 52.1 4.17 45.3 4.77 47.8 8.47 FX exp 92.8 6.96 109.8 8.19 72.3 4.00 48.4 4.84 48.8 4.73 42.0 4.72 Post FX d1 108.2 5.26 117.0 8.09 83.6 4.95 53.3 5.35 51.4 4.97 49.1 3.73 Post FX day 2 109.1 6.78 114.1 8.40 83.8 4.29 52.0 5.51 45.2 5.82 43.9 4.33 Post FX day3 109.1 8.84 118.4 7.12 82.0 4.48 61.6 5.47 67.2 5.34 70.0 9.28

253

~ 4 day 10 day 1M 3M 6M 1Y

N = 6-11 Mean

SEM

N = 9-10 Mean

SEM

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 4 Mean

SEM

N = 5-6 Mean

SEM

Episode duration Normal day in pen 11.6 0.57 17.9 1.18 24.8 2.23 25.0 1.76 28.0 3.63 Average of 3 or 4 days 12.7 0.96 17.5 0.91 22.4 1.33 24.3 1.75 27.3 3.16 Control exp 15.6 1.17 12.7 1.00 18.1 1.52 30.6 3.57 33.1 4.24 34.2 6.02 FX exp 16.4 1.27 14.1 1.48 20.6 1.22 33.8 4.09 30.3 3.00 36.5 4.17 Post FX d1 13.6 0.62 12.8 0.90 17.9 1.06 31.9 5.51 28.8 2.77 30.1 2.09 Post FX day 2 13.7 0.90 13.4 1.19 17.7 0.88 32.3 4.51 33.6 4.67 34.3 3.17 Post FX day3 14.2 1.44 12.6 0.79 18.2 1.15 25.4 2.25 21.9 2.01 22.3 2.70 Total activity score Normal day in pen 364101 24379 273966 22628 256500 20844 207330 27707 194202 19554 Average of 3 or 4 days 329552 29573 261100 17066 280306 18370 238344 27423 198719 17684 Control exp 231622 20094 353036 33624 276761 24624 193704 16161 143300 28081 119890 15178 FX exp 244185 27519 318034 29246 206102 16201 192362 25905 160958 29069 108207 10106 Post FX d1 273458 18561 346625 33925 262755 20552 217733 27649 179190 31005 136994 15370 Post FX day 2 286283 22827 336898 26493 258849 19842 203499 22156 142906 23973 115289 12053 Post FX day3 276287 25708 338189 28610 250920 17267 250660 18128 243123 24369 242252 36725

254

~ 4 day 10 day 1M 3M 6M 1Y

N = 6-11 Mean

SEM

N = 9-10 Mean

SEM

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 4 Mean

SEM

N = 5-6 Mean

SEM

% time mobile Normal day in pen 49.8 1.73 56.1 2.10 72.4 3.97 61.9 7.19 62.3 4.58 Average of 3 or 4 days 48.8 1.40 53.3 1.96 73.4 3.64 65.1 5.07 62.5 4.42 Control exp 42.5 1.72 51.3 1.75 57.2 1.44 64.5 4.20 53.1 6.23 46.5 5.11 FX exp 45.7 4.02 49.8 1.72 49.3 2.01 65.2 5.04 55.8 6.46 47.0 5.52 Post FX d1 43.7 1.58 50.9 1.88 54.6 2.47 68.5 3.69 60.1 7.42 50.1 6.64 Post FX day 2 45.2 1.48 51.3 0.98 53.6 2.76 66.9 3.71 54.5 3.87 47.3 5.76 Post FX day3 43.9 1.60 49.1 1.72 53.4 2.59 71.8 3.33 63.4 5.33 59.6 3.89 % time immobile Normal day in pen 50.3 1.73 43.9 2.10 27.6 3.97 38.2 7.19 37.7 4.58 Average of 3 or 4 days 51.2 1.40 46.7 1.96 26.6 3.64 34.9 5.07 37.5 4.42 Control exp 55.3 3.78 48.7 1.75 42.8 1.44 35.5 4.20 46.9 6.23 53.5 5.11 FX exp 55.4 2.98 50.2 1.72 50.7 2.01 34.8 5.04 44.2 6.46 53.0 5.52 Post FX d1 56.3 1.58 49.1 1.88 45.4 2.47 31.5 3.69 39.9 7.42 50.0 6.64 Post FX day 2 54.8 1.48 48.7 0.98 46.4 2.76 33.1 3.71 45.5 3.87 52.8 5.76 Post FX day3 56.1 1.60 50.9 1.72 46.6 2.59 28.2 3.33 36.6 5.33 40.4 3.89

255

Appendix 2: Heart Rate, Arterial Pressure And HRV In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection:

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. 2.1: Heart Rate And Arterial Pressure: ~4 day 10 day 1M 3M 6M 1Y

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 5-8 Mean

SEM

N = 12 Mean SEM

N = 7 Mean

SEM

N = 9 Mean SEM

Heart rate -30 min 228 9.5 203 5.7 241 8.9 129 7.6 108 5.6 104 4.7 -15 min 228 9.1 196 6.9 241 7.2 125 6.0 106 5.9 115 6.3 5 min 195 9.3 179 7.6 210 5.2 123 4.7 107 9.3 102 6.6 15min 197 9.0 180 8.9 213 5.0 115 4.5 104 6.6 103 8.1 30min 199 9.5 179 7.9 215 6.1 108 3.3 100 4.8 98 5.4 60min 209 7.9 200 6.4 221 5.8 108 4.9 92 2.6 89 2.8 120min 223 9.7 206 11.6 230 12.4 107 4.0 95 3.9 90 4.1 Arterial pressure -30 min 67.8 1.4 73.5 3.1 68.3 2.1 75.5 3.0 71.5 3.6 85.0 1.9 -15 min 66.6 1.8 75.2 3.4 67.5 2.3 75.1 2.2 70.3 2.8 84.3 2.2 5 min 72.4 1.4 85.5 2.6 72.8 2.0 82.8 3.0 79.9 4.6 97.6 3.2 15min 70.2 1.8 81.3 2.6 71.5 2.1 80.2 1.9 75.9 3.6 90.1 1.7 30min 67.8 1.4 77.2 2.2 67.9 1.6 79.5 2.0 73.3 3.8 89.8 1.7 60min 68.7 1.6 79.9 3.2 69.0 2.0 77.5 1.9 74.2 3.5 88.3 1.9 120min 68.7 1.8 76.5 2.2 71.2 2.2 75.5 2.7 74.3 3.6 87.1 2.3

256

2.2: Time Domain Of HRV (SDNN, SD Delta NN, RMSSD): 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. ~ 4 day 10 day 1M 3M 6M 1Y

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 5-8 Mean

SEM

N = 12 Mean SEM

N = 7 Mean

SEM

N = 9 Mean SEM

SDNN -30 min 20.6 2.5 29.2 2.6 51.6 12.9 61.1 6.2 67.5 5.7 112.1 15.9 30min 28.9 3.7 43.8 4.9 72.4 13.9 72.8 6.3 96.1 13.1 140.2 14.5 60min 24.0 3.1 38.0 2.3 55.1 11.5 76.6 7.6 82.4 11.8 123.7 16.4 90 min 23.5 3.4 34.7 2.2 51.9 9.5 74.6 7.4 78.5 7.4 118.4 18.5 120 min 18.4 2.8 36.4 5.5 38.6 5.6 66.6 6.6 75.8 10.1 116.0 17.2 SD Delta NN -30 min 16.8 2.8 18.0 2.6 37.0 12.1 51.9 8.2 49.8 11.0 91.0 12.4 30min 22.4 3.2 30.7 4.7 57.9 14.4 57.2 7.6 73.1 18.1 110.8 15.5 60min 22.0 3.1 22.5 2.0 41.0 11.8 72.6 14.5 53.4 16.2 85.7 12.5 90 min 21.5 4.7 20.1 2.3 37.2 9.5 68.1 11.2 55.9 10.4 81.9 13.8 120 min 14.6 2.9 29.6 10.1 23.4 6.4 59.0 6.8 59.8 11.7 87.1 13.4 RMSSD -30 min 16.8 2.8 18.0 2.6 36.9 12.1 51.9 8.1 49.8 11.0 91.0 12.4 30min 22.4 3.2 30.7 4.7 57.9 14.4 57.2 7.6 73.1 18.1 110.8 15.5 60min 22.0 3.1 22.5 2.0 41.0 11.8 72.6 14.5 53.4 16.2 85.7 12.5 90 min 21.5 4.7 20.1 2.3 37.2 9.5 68.1 11.2 55.9 10.4 81.9 13.8 120 min 14.6 2.9 29.6 10.1 23.4 6.4 59.0 6.8 59.8 11.7 87.1 13.4

257

2.3: Frequency Domain Of HRV (LF Power, HF Power, LF/HF Ratio): 1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. ~ 4 day 10 day 1M 3M 6M 1Y

N = 9-12 Mean

SEM

N = 12 Mean

SEM

N = 5-8 Mean

SEM

N = 12 Mean SEM

N = 7 Mean

SEM

N = 9 Mean SEM

LF power -30 min 75.2 17.4 192.5 29.8 711.4 282.2 531.1 90.4 727.7 129.8 2410.1 665.4 30min 104.7 27.1 275.8 35.0 882.6 275.4 664.6 110.9 1250.7 524.0 3614.1 765.6 60min 94.2 21.5 263.3 43.1 642.3 203.3 675.4 118.2 1087.2 460.5 2150.2 531.9 90 min 73.3 15.9 261.6 57.5 500.3 111.7 632.4 92.8 944.8 241.3 2218.2 556.5 120 min 67.7 17.4 245.0 45.3 337.2 78.7 671.1 103.9 1094.7 306.5 2749.8 632.0 HF power -30 min 47.7 10.6 74.1 10.8 532.8 288.6 459.0 104.2 570.4 317.4 2176.1 535.9 30min 55.5 14.8 131.0 29.3 865.9 383.3 598.1 140.1 954.1 654.0 3585.3 866.0 60min 60.6 11.0 99.6 15.0 446.5 200.6 662.8 147.3 732.0 483.7 1950.0 521.5 90 min 56.7 15.7 89.5 18.8 310.9 130.3 618.1 124.1 653.9 336.5 1996.7 605.1 120 min 37.8 11.5 134.0 53.6 115.8 19.9 629.1 117.4 956.4 527.0 2478.0 630.2 LF/HF ratio -30 min 1.7 0.2 3.1 0.5 2.9 0.6 1.5 0.2 2.2 0.4 1.2 0.2 30min 2.2 0.4 2.9 0.5 2.1 0.5 1.5 0.2 2.6 0.7 1.2 0.2 60min 1.6 0.2 3.0 0.4 2.0 0.4 1.4 0.3 3.5 1.0 1.3 0.1 90 min 1.5 0.2 3.3 0.4 2.8 0.6 1.4 0.2 2.5 0.6 1.4 0.2 120 min 2.0 0.3 3.1 0.6 3.5 1.0 1.4 0.2 2.5 0.9 1.4 0.2

258

Appendix 3: Arterial Blood Gases And Body Temperature In The Lambs At ~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection:

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. ~4 day 10 day 1M 3M 6M 1Y

N = 11 N = 8-12 N = 6-10 N = 10-12 N = 7 N = 10

pH Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM -15 min 7.385 0.010 7.433 0.009 7.443 0.010 7.496 0.014 7.502 0.019 7.482 0.014 5 min 7.369 0.013 7.427 0.011 7.439 0.007 7.494 0.011 7.503 0.013 7.498 0.012 15 min 7.388 0.010 7.425 0.008 7.439 0.011 7.475 0.012 7.481 0.008 7.506 0.011 30 min 7.383 0.009 7.424 0.009 7.428 0.006 7.489 0.009 7.487 0.014 7.506 0.011 60 min 7.378 0.012 7.411 0.006 7.434 0.012 7.467 0.016 7.474 0.011 7.488 0.008 120 min 7.375 0.010 7.413 0.007 7.456 0.009 7.467 0.014 7.479 0.016 7.488 0.018 Temperature -15 min 39.8 0.1 39.6 0.2 39.7 0.2 39.6 0.1 39.6 0.1 39.3 0.2 5 min 39.5 0.2 39.7 0.2 39.5 0.1 39.7 0.1 39.6 0.1 39.3 0.2 15 min 39.3 0.3 39.6 0.2 39.6 0.1 39.7 0.1 39.5 0.1 39.4 0.1 30 min 39.4 0.2 39.9 0.2 39.7 0.1 39.8 0.1 39.5 0.1 39.2 0.2 60 min 39.4 0.2 39.9 0.2 39.7 0.1 40 0.2 39.5 0.1 39.3 0.1 120 min 39.5 0.2 39.7 0.1 39.5 0.2 39.9 0.1 39.4 0.1 39.3 0.1 O2 saturation -15 min 93.3 0.4 93.1 0.5 93.2 0.3 95.4 0.2 94.5 1.4 95.0 0.4 5 min 93.4 0.4 92.5 0.7 89.0 1.5 93.8 1.1 92.1 1.9 92.9 0.8 15 min 93.5 0.4 92.8 0.4 93.1 0.4 95.3 0.4 93.6 1.4 96.0 0.2 30 min 93.5 0.4 92.9 0.6 92.3 0.4 94.6 0.6 94.4 0.9 95.5 0.3 60 min 93.2 0.5 92.4 0.3 93.4 0.3 94.6 0.3 94.9 0.5 95.2 0.2 120 min 92.9 1.0 93.0 0.3 93.0 1.0 95.2 0.2 95.1 0.4 95.7 0.3

259

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. ~4 day 10 day 1M 3M 6M 1Y

N = 11 N = 8-12 N = 6-10 N = 10-12 N = 7 N = 10

PO2 Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM -15 min 88.0 3.8 95.0 3.9 95.3 2.5 104.7 2.9 111.9 5.4 110.5 2.5 5 min 89.6 2.8 93.7 3.2 81.0 5.0 96.2 3.5 95.0 7.7 94.0 5.8 15min 90.5 4.0 94.0 2.7 93.5 2.2 103.0 3.5 104.1 6.5 114.3 2.1 30min 86.9 3.4 94.4 4.0 92.6 1.8 99.9 3.4 106.6 5.5 108.6 3.2 60min 89.7 4.2 95.2 2.4 95.5 2.1 102.1 2.5 108.3 2.1 107.1 2.6 120min 91.9 3.5 96.7 1.7 94.4 3.3 103.4 2.4 106.3 3.7 111.8 2.1 PCO2

-15 min 42.2 1.0 40.9 0.7 41.5 1.2 36.6 1.2 36.2 1.2 37.6 1.1 5 min 42.8 1.6 41.7 1.1 40.8 1.0 36.8 0.7 36.6 0.9 36.4 0.8 15min 40.9 0.9 41.5 0.7 40.3 1.0 38.2 1.3 38.0 0.7 35.5 0.8 30min 40.9 0.7 41.9 0.9 41.4 0.8 37.9 0.6 37.7 0.9 36.0 0.9 60min 40.0 1.1 41.2 0.7 41.1 1.7 38.9 1.4 36.8 0.6 36.7 0.7 120min 39.3 0.8 40.9 0.6 39.1 1.0 38.5 1.6 37.2 0.3 36.1 1.2

260

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year.

~4 day 10 day 1M 3M 6M 1Y N = 11 N = 8-12 N = 6-10 N = 10-12 N = 7 N = 10

Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEMBE -15 min 0.27 0.75 3.09 0.62 4.18 0.60 5.17 0.81 5.43 1.59 4.70 0.62 5 min -0.73 0.73 3.00 0.47 3.55 0.68 5.17 0.89 5.86 1.03 5.10 0.57 15min -0.45 0.72 2.91 0.46 3.00 0.56 4.64 0.91 4.86 0.88 5.00 0.68 30min -0.73 0.71 2.91 0.49 3.00 0.45 5.58 0.92 5.14 1.20 5.60 0.65 60min -1.55 0.89 1.64 0.43 3.18 0.60 4.45 0.96 3.71 0.92 4.40 0.73 120min -2.09 0.51 1.64 0.49 3.82 0.75 4.00 0.93 4.43 1.23 4.00 0.86 HCO3 -15 min 24.6 0.7 26.7 0.5 27.5 0.6 27.6 0.7 27.9 1.3 27.6 0.5 5 min 24.1 0.6 26.8 0.4 26.9 0.7 27.8 0.7 28.2 0.8 27.7 0.4 15min 23.9 0.6 26.6 0.4 26.5 0.5 27.5 0.8 27.9 0.8 27.5 0.5 30min 23.8 0.6 26.7 0.4 26.6 0.5 28.2 0.8 28.0 1.0 28.0 0.6 60min 23.1 0.7 25.5 0.4 26.6 0.6 27.4 0.8 26.5 0.7 27.4 0.6 120min 22.5 0.4 25.4 0.4 26.8 0.7 27.1 0.8 27.2 0.9 26.8 0.6 Hb -15 min 11.2 0.4 9.0 0.4 8.2 0.6 9.6 0.2 9.2 0.2 9.6 0.2 5 min 11.3 0.4 9.1 0.4 8.9 0.6 9.9 0.2 9.8 0.2 10.6 0.2 15min 11.3 0.4 9.1 0.4 8.9 0.7 9.9 0.2 9.6 0.2 10.0 0.2 30min 11.3 0.4 9.2 0.4 8.8 0.7 9.7 0.2 9.3 0.2 9.8 0.2 60min 11.3 0.5 9.2 0.4 8.7 0.7 9.8 0.2 9.3 0.2 9.7 0.2 120min 11.3 0.4 9.2 0.5 8.5 0.7 9.7 0.2 9.4 0.2 9.9 0.2

261

Appendix 4: Glucose, Lactate Concentration And Electrolite In The Lambs At~ 4, 10 Days, 1, 3, 6 And 12 Months Of Age With FX Injection:

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year. Mean ~4 day 10 day 1M 3M 6M 1Y N = 11 N = 8-12 N = 6-10 N = 10-12 N = 7 N = 10

Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Glucose -15 min 5.492 0.249 5.446 0.158 4.155 0.126 3.166 0.137 2.950 0.135 2.936 0.103 5 min 5.539 0.213 5.451 0.127 4.049 0.143 3.099 0.137 3.047 0.119 2.818 0.085 15min 5.345 0.214 5.321 0.138 4.020 0.138 3.166 0.158 2.981 0.087 2.894 0.087 30min 5.483 0.214 5.450 0.172 4.321 0.189 3.232 0.137 3.063 0.093 2.974 0.097 60min 5.667 0.194 5.590 0.105 4.294 0.172 3.257 0.160 3.139 0.080 3.026 0.113 120min 5.574 0.174 5.791 0.104 4.474 0.213 3.413 0.169 3.242 0.090 3.156 0.130 Lactate -15 min 0.904 0.054 0.947 0.225 0.820 0.275 0.633 0.063 0.510 0.043 0.562 0.044 5 min 0.909 0.062 0.811 0.112 0.859 0.300 0.645 0.063 0.595 0.063 0.580 0.039 15min 0.905 0.071 0.834 0.106 0.806 0.230 0.625 0.057 0.669 0.090 0.571 0.046 30min 0.841 0.058 0.651 0.062 0.820 0.221 0.665 0.077 0.650 0.099 0.547 0.039 60min 0.814 0.053 0.862 0.190 0.841 0.261 0.728 0.101 0.662 0.098 0.606 0.041 120min 0.784 0.069 0.777 0.090 0.821 0.237 0.619 0.078 0.540 0.042 0.550 0.043

262

1M = 1 month, 3M = 3 months, 6M = 6 months, 1Y = 1 year.

Mean ~4 day 10 day 1M 3M 6M 1Y N = 11 N = 8-12 N = 6-10 N = 10-12 N = 7 N = 10

Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Na+ -15 min 141 0.5 143 0.6 143 0.4  146 0.5 146 0.5 147 0.4

5 min 140 0.6 143 0.5 143 0.5  146 0.4 147 0.5 147 0.4

15min 141 0.5 143 0.4 143 0.4  146 0.4 147 0.5 147 0.4

30min 141 0.6 143 0.4 143 0.4  146 0.4 146 0.6 147 0.5

60min 140 0.5 143 0.4 143 0.4  145 0.4 146 0.9 147 0.4

120min 140 0.7 143 0.5 143 0.5  145 0.4 146 0.8 146 0.3

K+ -15 min 4.05 0.12 3.85 0.11 4.09 0.11  4.02 0.09 3.83 0.10 4.01 0.08

5 min 4.29 0.14 3.89 0.11 4.12 0.11  4.18 0.10 3.96 0.10 4.06 0.05

15min 4.27 0.15 3.85 0.12 4.17 0.13  4.06 0.08 3.84 0.13 3.95 0.04

30min 4.28 0.13 3.99 0.14 4.14 0.09  4.13 0.07 3.81 0.10 3.95 0.05

60min 4.33 0.11 3.99 0.14 4.09 0.10  4.31 0.07 3.96 0.16 4.02 0.08

120min 4.40 0.13 4.02 0.14 4.12 0.13  4.31 0.12 4.17 0.09 4.19 0.07

iCa -15 min 1.51 0.03 1.48 0.02 1.42 0.02  1.22 0.02 1.23 0.04 1.20 0.02

5 min 1.49 0.03 1.48 0.02 1.44 0.01  1.23 0.02 1.23 0.03 1.20 0.02

15min 1.48 0.03 1.47 0.02 1.41 0.01  1.21 0.02 1.21 0.02 1.17 0.02

30min 1.52 0.03 1.49 0.02 1.43 0.01  1.22 0.02 1.20 0.02 1.17 0.02

60min 1.51 0.03 1.47 0.02 1.43 0.01  1.25 0.03 1.25 0.03 1.20 0.02

120min 1.50 0.03 1.47 0.01 1.40 0.01  1.28 0.02 1.28 0.03 1.23 0.02