Placental insufficiency resulting in fetal growth restriction alters synaptic development and

217 Pages·2017·13.19 MB·English

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Karolina Piorkowska, The University of Western Ontario
Supervisor: Bryan Richardson, The University of Western Ontario
A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in
Physiology
© Karolina Piorkowska 2012
Follow this and additional works at: https://ir.lib.uwo.ca/etd
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Piorkowska, Karolina, "Placental insufficiency resulting in fetal growth restriction alters synaptic
development and neuronal myelination in guinea pigs at term" (2012). Electronic Thesis and Dissertation
Repository. 615.
https://ir.lib.uwo.ca/etd/615
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PLACENTAL INSUFFICIENCY RESULTING IN FETAL GROWTH RESTRICTION
ALTERS SYNAPTIC DEVELOPMENT AND NEURONAL MYELINATION IN
GUINEA PIGS AT TERM
(SPINE TITLE: FETAL GROWTH RESTRICTION AND GUINEA PIG BRAIN
DEVELOPMENT)
(THESIS FORMAT: INTEGRATED-ARTICLE)
by
Karolina Piorkowska BMSc
Graduate Program in Physiology- Developmental Biology

Submitted in partial fulfillment
of the requirements for the degree
Master of Science

The School of Graduate and Postdoctoral Studies
The University of Western Ontario
London, Ontario, Canada

© Karolina Piorkowska 2012
THE UNIVERSITY OF WESTERN ONTARIO
School of Graduate and Postdoctoral Studies

CERTIFICATE OF EXAMINATION

Supervisor Examiners

______________________________ ______________________________
Dr. Bryan Richardson Dr. David Lee

Supervisory Committee
______________________________
Dr. Stan Leung
______________________________
Dr. Marco Prado
______________________________
Dr. Daniel Hardy
______________________________
Dr. Robert Hammond

______________________________
Dr. John Ciriello

The thesis by

Karolina Piorkowska

entitled:
Placental Insufficiency resulting in Fetal Growth Restriction Alters Synaptic Development
and Neuronal Myelination in Guinea Pigs at Term
is accepted in partial fulfillment of the
requirements for the degree of
Masters of Science

______________________ _______________________________
Date Chair of the Thesis Examination Board

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ABSTRACT
Aberrant neuronal connectivity in utero may underlie the association between fetal
growth restriction (FGR) and increased risk for later cognitive disorders and encephalopathy.
This study examines changes in synaptic development and myelination focussing on the
hippocampus using a guinea pig model of placental insufficiency. Placental insufficiency was
induced at mid-gestation by uterine artery ligation or cauterization which produced fetuses with a
range of body weight and proportion at term. Synaptic markers, synaptophysin and
synaptopodin, were decreased in FGR animals suggesting fewer synapses were formed and
furthermore that fewer synapses matured with symmetrical growth restriction when compared to
appropriate for gestational age guinea pigs. Myelination, measured using myelin basic protein
and luxol fast blue, was also reduced in FGR animals and increased in large animals compared to
appropriate size for gestational age guinea pigs. Therefore, neuronal connections altered with
FGR and may spur difficulties in early childhood neurodevelopment and various neurological
sequellae.
KEYWORDS
Fetal growth restriction, brain development, guinea pig, synaptogenesis, myelination,
synaptophysin, synaptopodin, myelin basic protein, luxol fast blue, hippocampus

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CO-AUTHORSHIP
The following people contributed to the manuscripts contained within this thesis in the following
ways:
Dr. B Richardson: Supervisor throughout all projects, provided grant funding to complete
manuscripts, edited manuscript
J Thompson: Performed animal surgery and necropsy, edited manuscripts
K Nygard: Provided technical support for histology and image analysis, input into
data analysis
B Matushewski: Performed animal surgery and necropsy
Dr. M Prado: Graduate Studies representative and member of advisory committee,
provided support and advice throughout the project
Dr. J Ciriello: Member of advisory committee, provided support and advice throughout
the project
Dr. R Hammond: Member of advisory committee, provided neuroanatomy and technical
support for histology and image analysis, provided support and advice
throughout the project

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Don’t be afraid to take a big step if one is indicated. You can’t cross a chiasm in two small jumps
(…without extensive planning, creativity, and building materials -Karolina Piorkowska)
-David Lloyd George

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To everyone who has supported me
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TABLE OF CONTENTS
Page
CERTIFICATE OF EXAMINATION ii
ABSTRACT AND KEYWORDS iii
CO-AUTHORSHIP iv
EPIGRAPH v
DEDICATION vi
TABLE OF CONTENTS vi
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS AND SYMBOLS xiii
CHAPTER 1 - LITERATURE REVIEW 1
1.1 Clinical Relevance 2
1.1.1 Incidence and classification of FGR 2
1.1.2 Etiology of FGR 5
1.1.2.1 Placental insufficiency 6
1.1.2.2 Developmental origin of symmetrical FGR 8
1.1.2.3 Developmental origin of asymmetrical FGR 9
1.1.3 FGR and life-long health 10
1.1.4 FGR and neurological outcome 13
1.2 Fetal Brain Growth and Development 15
1.2.1 Synaptogenesis 16
1.2.1.1 Protein markers of synapse development 19
1.2.2 Myelination 21
1.2.2.1 Markers for myelin development 23
1.2.3 Development and function of the hippocampus 24
1.3 Animal Models of FGR and Adverse Development 25
1.3.1 Guinea pig brain development 25
1.3.2 Animal studies of FGR with placental insufficiency 26
1.3.3 Animal studies of FGR and structural brain changes 28
1.3.4 Animal studies of FGR and neurobehavioural outcomes 29
1.4 Summary 30
1.5 References 32
CHAPTER 2 - RATIONALE, HYPOTHESIS AND RESEARCH OBJECTIVES 46
2.1 Rationale 47
2.2 Hypotheses 49
2.3 Objectives 50
2.4 References 52
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CHAPTER 3 FETAL GUINEA PIGS WITH ALTERED GROWTH AT TERM
AFTER MID-GESTATION LIGATION OR ABLATION OF
UTERINE VESSELS 55
3.1 Introduction 56
3.2 Materials and Methods 61
3.2.1 Surgical preparation 61
3.2.2 Surgical procedure and post-operative care 61
3.2.3 Necropsy and organ collection 62
3.2.4 Data acquisition and statistical analysis 63
3.3 Results 67
3.3.1 Generating fetal growth restriction 67
3.3.2 Fetal cohort characteristic data 68
3.3.3 Fetal cohort organ weights 69
3.4 Discussion 89
3.5 Conclusion 94
3.6 References 96
CHAPTER 4 THE IMPACT OF FETAL GROWTH RESTRICTION ON
SYNPASE FORMATION AND MATURATION IN THE
GUINEA PIG BRAIN AT TERM 100
4.1 Introduction 101
4.2 Materials and Methods 105
4.2.1 Surgical preparation and necropsy 105
4.2.2 Fetal guinea pig cohorts and brain processing for histology 105
4.2.3 Fetal guinea pig brain processing for Western blots 106
4.2.4 Immunohistochemistry of synaptophysin 107
4.2.5 Immunofluorescence of synaptopodin 108
4.2.6 Imaging and statistical analysis of synaptophysin and synaptopodin 109
4.2.7 Western blot protocol for synaptophysin and synaptopodin 112
4.3 Results 126
4.3.1 Synaptophysin quantification 126
4.3.2 Synaptopodin quantification 126
4.3.3 Western blot analysis for synaptophysin and synaptopodin 127
4.4 Discussion 138
4.5 Conclusion 142
4.6 References 143
CHAPTER 5 EFFECT OF FETAL GROWTH RESTRICTION ON
MYELIN FORMATION IN THE GUINEA PIG BRAIN AT
TERM 150
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5.1 Introduction 151
5.2 Materials and Methods 154
5.2.1 Fetal guinea pig collection and brain processing for histology
and Western blots 154
5.2.2 Immunohistochemistry of myelin basic protein 155
5.2.3 Histochemical stain for myelin: luxol fast blue 155
5.2.4 Imaging and statistical analysis for myelin basic protein and luxol
fast blue 156
5.2.5 Western blot protocol for myelin basic protein 158
5.3 Results 164
5.3.1 Myelin basic protein quantification 164
5.3.2 Luxol fast blue quantification 164
5.3.3 Western blot analysis for myelin basic protein 165
5.4 Discussion 176
5.5 Conclusion 180
5.6 References 181
CHAPTER 6 GENERAL DISCUSSION 185
6.1. General discussion 186
6.2. Future studies 192
6.3. Conclusions 193
6.4. References 195
CURRICULUM VITAE 200

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