expression of gaba receptors in human scleraeprints.qut.edu.au/68604/2/emily_mcdonald_thesis.pdf ·...

Emily F. McDonald: GABA Receptors in Human Sclera

Expression of GABA Receptors in Human Sclera

Emily McDonald (nee Shanahan)

BSc (BmedSc), Hons (Microbiol)

The School of Optometry and Vision Science, Faculty of Health,

Vision Improvement Domain, Institute of Health and Biomedical

Innovation,

Queensland University of Technology

A thesis submitted in fulfilment of the requirements for the

Degree of Master of Applied Science (Research)

2014


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Keywords

Choroid

Cultured Human Scleral Cells

Eye

Flow Cytometry

Gamma‐Aminobutyric Acid (GABA)

GABA Receptors

Immunohistochemistry

mRNA

Reverse‐Transcriptase Polymerase Chain Reaction

Retina

Retinal Pigment Epithelium

Sclera


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Abstract

Background: Myopia, colloquially known as short‐sightedness, is a common refractive error

and is caused by elongation of the globe, i.e. axial elongation. Myopia, although

predominantly caused by excessive elongation, can be associated with optical alterations.

The sclera must expand to allow for this increase in size and, ultimately, must grow, or

remodel, for myopia to develop. It has been found that ‐aminobutyric acid (GABA), a

retinal neurotransmitter, influences eye growth and, accordingly, refractive development. In

the chick model of myopia, GABA antagonists have been shown to inhibit experimental

myopia and have been proposed as having a promising clinical role in the myopia treatment.

Although the mechanism for this effect is unknown, it has been recently shown that GABA

receptors are located in chick sclera, cornea and retinal pigment epithelium (RPE). It is well

known that there are GABA receptors present in the retina, including that of humans, and

that GABA is used as a retinal transmitter; however, not much is known about GABA in

other ocular tissues. The aim of this study was to determine the presence or absence of

GABA receptors in human scleral tissue and cultured human scleral cells, as well as

choroidal/RPE and retinal tissue.

Methods: Human ocular tissues were obtained from six donor eyes from the Queensland Eye

Bank. Three linked experiments were undertaken (i) immunohistochemistry (IHC) on

human scleral sections, (ii) reverse‐transcriptase polymerase chain reaction (RT‐PCR) of

ocular tissues, and (iii) RT‐PCR and flow cytometry (Dominici et al.) of cultured scleral cells.

(i) IHC was undertaken on human scleral tissue from one donor using antibodies raised

against three different GABA receptor subunits previously shown to be expressed in human

retinal tissue – GABAA Alpha 1, GABAC Rho 1, and GABAC Rho 2. As representative of the

GABAB receptor, the following antibody was also included: GABAB R2. The antibodies were

applied to the scleral sections taken from the four quadrants of the eye – superior, inferior,

medial, and lateral. The tissue required bleaching due to the presence of melanocytes. (ii)

Following isolation of total ribonucleic acid (RNA), RT‐PCR was undertaken to determine

gene expression of all known GABA receptor subunits on human scleral tissue (n=3 eyes),

choroid/RPE tissue (n=2 eyes), and retinal tissue (n=2 eyes). (iii) Scleral cells were cultured

using standard techniques utilising DMEM and FBS and passaged three times. RT‐PCR was

performed (n=2 eyes) and scleral cells were subsequently characterised using FC; the cells

were labelled with cell surface markers CD31, CD34, CD44, CD45, CD73, CD90, CD105,

GABAA‐α1, GABAB‐2, GABR‐R1, and GABR‐R2.

Results: Findings from the IHC of the scleral sections (i) were inconclusive due to

background staining. This may be due, at least in part, to the bleaching step (necessary due

to the presence of melanocytes) which increased the basophilia of the tissue. An alternative

strategy was to look at which GABA receptor genes were expressed. The gene expression

component of the project (ii) revealed the presence of several GABA receptor subunits in all

of the ocular tissues tested, including the cultured scleral cells (iii). The three scleral donors

yielded positive RNA results for GABA subunits representing GABAA and GABAC receptors

using RT‐PCR. The cultured scleral cells similarly revealed the presence of GABAA and

GABAC receptor subunits and were additionally positive for GABAB. The choroid/RPE and

retinal samples were also positive for GABAA and GABAC receptor subunits; one of the

retinal donors also yielded a positive result for GABAB. FC showed that the scleral‐derived

cells grown as fibroblasts were mesenchymal stromal cells.


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Significance: This study is the first to show that GABA receptor gene expression was

observed in human sclera, human cultured mesenchymal stromal cells, and the combined

choroid/RPE. This finding has important implications for future research, including the role

of GABA in refractive development and myopia. Knowledge of the complete profile of

GABA receptor expression would identify which might be more specific targets for

treatment of myopia. Furthermore, other ocular functions mediated by these receptors are

yet to be determined.


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TABLE OF CONTENTS

KEYWORDS .................................................................................................................................... II

ABSTRACT..................................................................................................................................... III

LIST OF TABLES ....................................................................................................................... VIII

LIST OF FIGURES .......................................................................................................................... X

ABBREVIATIONS, ACRONYMS, & SYMBOLS ................................................................. XII

STATEMENT OF ORIGINAL AUTHORSHIP .................................................................... XIV

STRUCTURE OF THESIS ......................................................................................................... XIV

ACKNOWLEDGEMENTS ........................................................................................................ XV

1 LITERATURE REVIEW ............................................................................................................ 1

1.1 INTRODUCTION ............................................................................................................................................ 1

1.2 BRIEF OVERVIEW OF THE EYE ....................................................................................................................... 3

1.3 MYOPIA: DEFINITION, EPIDEMIOLOGY, & POSSIBLE CAUSES ..................................................................... 4

1.4 THE PROPOSED MYOPIA SIGNALLING CASCADE: WHERE THE SCLERA IS THOUGHT TO FIT IN ................ 5

1.4.1 Neural Retina: Generates Primary Signal for Myopia Development? .................................... 6

1.4.2 Retinal Pigment Epithelium: Signal Relay in the Control of Eye Growth? .............................. 8

1.4.3 Choroid: Responsible for Fluid Regulation ............................................................................ 10

1.4.4 Sclera: Responsible for Controlling Eye Dimensions ............................................................. 11

1.5 ‐AMINOBUTYRIC ACID ............................................................................................................................. 11

1.5.1 ‐Aminobutyric Acid Receptors – GABAA, GABAB, and GABAC .............................................. 12

1.5.2 ‐Aminobutyric Acid as a Piece in the Myopia Puzzle: GABA in Eye Growth ........................ 17

1.5.3 ‐Aminobutyric Acid within the Human Eye ......................................................................... 17

1.5.4 ‐Aminobutyric Acid in Animal Models ................................................................................. 18

1.5.5 ‐Aminobutyric Acid in Fibroblasts of other Tissues ............................................................. 21

1.5.6 Pharmacological Factors Affecting Eye Growth in Animal Models ...................................... 22

1.6 HUMAN SCLERA ......................................................................................................................................... 22

1.6.1 Gross Structure & Function of Sclera .................................................................................... 23

1.6.2 Composition of the Sclera ..................................................................................................... 24

1.6.3 Collagen ................................................................................................................................ 26

1.6.4 Elastin ................................................................................................................................... 26

1.6.5 Enzymes ................................................................................................................................ 26

1.6.6 Scleral Fibroblasts ................................................................................................................. 29

1.6.7 Glycoproteins ........................................................................................................................ 30

1.6.8 Growth Factors ..................................................................................................................... 30


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1.6.9 Proteoglycans ....................................................................................................................... 30

1.6.10 Other Receptor Types known to be Present in Human Sclera ............................................ 32

1.6.11 Factors Affecting Human Scleral Structure & Function ...................................................... 34

1.7 SUMMARY ................................................................................................................................................... 35

1.8 RESEARCH AIMS ......................................................................................................................................... 37

2 MATERIALS AND METHODOLOGY ............................................................................... 38

2.1 HUMAN RESEARCH ETHICS ISSUES ............................................................................................................ 38

2.2 EYE COLLECTION ....................................................................................................................................... 38

2.3 PREPARATION FOR MICROTOMY ............................................................................................................... 39

2.4 MICROTOMY ............................................................................................................................................... 40

2.5 PRELIMINARY WORK: H&E AND BLEACHING OPTIMISATION ................................................................. 40

2.6 IMMUNOHISTOCHEMISTRY: EXPERIMENT #1 ............................................................................................. 43

2.7 CELL CULTURING & FLOW CYTOMETRY: EXPERIMENT #2 ........................................................................ 43

2.8 ANALYSIS OF GABA RECEPTOR GENE EXPRESSION IN OCULAR TISSUES AND CELLS: EXPERIMENT #3. 44

2.8.1 Primer Design ....................................................................................................................... 44

2.8.2 RNA Isolation and Reverse‐Transcription ............................................................................. 47

2.8.3 RT‐PCR Amplification of GABA Receptors ............................................................................. 49

3 RESULTS ................................................................................................................................... 51

3.1 IMMUNOHISTOCHEMISTRY: EXPERIMENT #1 ............................................................................................. 51

3.2 FLOW CYTOMETRY: EXPERIMENT #2 .......................................................................................................... 53

3.3 ANALYSIS OF GABA RECEPTOR GENE EXPRESSION IN OCULAR TISSUES AND CELLS: EXPERIMENT #3. 56

3.3.1 RT‐PCR Amplification of GABA Receptors ............................................................................. 56

4 DISCUSSION ........................................................................................................................... 60

4.1 SUMMARY OF FINDINGS ............................................................................................................................. 60

4.2 LIMITATIONS OF IMMUNOHISTOCHEMISTRY ............................................................................................. 60

4.3 POLYMERASE CHAIN REACTION METHODOLOGICAL ISSUES ................................................................... 61

4.4 GABA RECEPTORS IN SCLERA ................................................................................................................... 61

4.5 GABA RECEPTORS IN RETINAL PIGMENT EPITHELIUM & CHOROID ....................................................... 62

4.6 GABA RECEPTORS IN RETINA ................................................................................................................... 62

4.7 LIMITATIONS OF FLOW CYTOMETRY.......................................................................................................... 63

4.8 RELEVANCE TO MYOPIA ............................................................................................................................ 63

4.9 LIMITATIONS .............................................................................................................................................. 64

4.10 REAL WORLD IMPLICATIONS ................................................................................................................... 65

4.11 FUTURE DIRECTIONS ................................................................................................................................ 65

4.12 CONCLUSION ............................................................................................................................................ 67


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REFERENCES/BIBLIOGRAPHY ................................................................................................ 68

APPENDIX ONE: ANIMAL STUDIES – BACKGROUND .................................................. 90

APPENDIX TWO: GROSS ANATOMY OF HUMAN SCLERA .......................................... 92

APPENDIX THREE: GEL RESULTS.......................................................................................... 95

APPENDIX FOUR: GABA RECEPTOR GENE SEQUENCING ......................................... 129

APPENDIX FIVE: GENOME MAPS ........................................................................................ 136


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LIST OF TABLES

Table 1.1: Known functions of the RPE as compiled by Strauss in 2005 (Strauss, 2005).......... . 9

Table 1.2: Characteristics of the three GABA receptor types: GABAA, GABAB, and GABAC.. 16

Table 1.3: Summaries of notable papers utilising GABA in animal studies......... ..................... 17

Table 1.4: Ocular tissues – human and animal – found to have GABA receptors present...... 20

Table 1.5: Main biochemical constituents present in human sclera......... ................................... 25

Table 1.6: Known functions of MMPs......... .................................................................................... 27

Table 1.7: Known MMPs and their associated substrate specificity......... .................................. 28

Table 1.8: Receptor types observed in human sclera.......... .......................................................... 33

Table 1.9: Location of muscarinic receptors in ocular and non‐ocular tissues. ......................... 33

Table 1.10: Key functions of receptors present in human sclera. ................................................ 34

Table 2.1: Donor characteristics; information supplied from the QEB. ..................................... 38

Table 2.2: Individual primers (and expected product size) for each gene, including all known

transcript variants where applicable and represented by a ʹVʹ in the gene name,

representing a GABA receptor subunit. .................................................................................. 46

Table 2.3: Annealing temperatures for primers following exposure of all cells and tissues to

three different annealing ranges. ............................................................................................. 47

Table 2.4: Results from NanoDrop. ................................................................................................. 49

Table 3.1: Raw data analysis obtained from FC for human scleral stromal cells. .................... 55

Table 3.2: The results for each of the GABA receptor subunit genes as demonstrated by PCR.

....................................................................................................................................................... 58

Table A1: Recent examples of animal models used for myopia research. ................................. 91

Table A2: RT‐PCR results for mesenchymal stromal cells obtained from donor 6952. ........... 95



Table A5: RT‐PCR results for mesenchymal stromal cells obtained from donors 6952 & 6953.

..................................................................................................................................................... 101

Table A6: RT‐PCR results for mesenchymal stromal cells obtained from donor 6953. ......... 103

Table A7: RT‐PCR results for choroid/RPE, retina, and mesenchymal stromal cells obtained

from donors 6952 & 6953. ........................................................................................................ 104


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Table A8: RT‐PCR results for choroid/RPE, retina, sclera, and mesenchymal stromal cells

obtained from donors 6952 & 6953. ....................................................................................... 106

Table A9: RT‐PCR results for sclera obtained from donor 6952. .............................................. 108

Table A10: RT‐PCR results for sclera obtained from donor 6953. ............................................ 109

Table A11: RT‐PCR results for sclera obtained from donor 6954. ............................................ 110

Table A12: RT‐PCR results for sclera obtained from donors 6952, 6953, & 6954. ................... 111

Table A13: RT‐PCR results for sclera obtained from donors 6952, 6953, & 6954. ................... 113

Table A14: RT‐PCR results for choroid/RPE obtained from donor 6952. ................................ 115

Table A15: RT‐PCR results for choroid/RPE & retina obtained from donors 6952 & 6953. .. 116

Table A16: RT‐PCR results for retina obtained from donor 6953. ............................................ 118

Table A17: RT‐PCR results for retina, choroid/RPE, sclera, and mesenchymal stromal cells

obtained from donor 6952. ...................................................................................................... 119

Table A18: RT‐PCR results for choroid/RPE ‐ donor 6953 ‐ & retina ‐ donor 6952. ............... 121

Table A19: RT‐PCR results for choroid/RPE & retina obtained from donors 6952 & 6953. .. 123

Table A20: RT‐PCR results for retina, choroid/RPE, sclera, mesenchymal stromal cells

obtained from donor 6952. ...................................................................................................... 125

Table A21: RT‐PCR results for mesenchymal stromal cells obtained from donor 6953. ....... 127

Table A22: RT‐PCR results for mesenchymal stromal cells obtained from donor 6952. ....... 128

Table A23: Relevant Blast results obtained from the AGRF for each product representative of

potential positive results for GABA receptor genes. ........................................................... 130


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LIST OF FIGUREs

Figure 1.1: Leading causes of visual loss in Australia (Baird et al., 2010).. .................................. 1

Figure 1.2: A pictorial representation of the human eye (Atchison and Smith, 2000) ................ 4

Figure 1.3: An emmetropisation model depicting the visual signalling cascade (Young, 2010)

......................................................................................................................................................... 6

Figure 1.4: The different layers of the retina and the cells associated with each of these layers

(Yang, 2004) ................................................................................................................................... 7

Figure 1.5: Chemical structure of ‐aminobutyric acid (Ng et al., 2011) .................................... 11

Figure 1.6: Structure of the GABAA receptor. ................................................................................. 13

Figure 1.7: Structure of the GABAB receptor (Enna, 2007) ........................................................... 14

Figure 1.8: Pharmacological differences between GABAA and GABAC receptors (Gottesmann,

2002, Bormann, 2000) ................................................................................................................. 15

Figure 1.9: Model illustrating the likely molecular structure – based on current knowledge –

of the ECM of the mammalian sclera (McBrien and Gentle, 2003) ...................................... 24

Figure 1.10: Proposed model of the role of scleral myofibroblast cells in the biochemical and

biomechanical remodelling that facilitates myopia development (McBrien et al., 2009). 29

Figure 1.11: Diagrammatic representation of the scleral remodelling that underpins myopia

development (McBrien et al., 2009). ......................................................................................... 35

Figure 2.1: Photograph (taken by EFM) of a typical eye specimen showing length of the optic

nerve and visible veins .............................................................................................................. 39

Figure 2.2: Scleral map highlighting superior, inferior, nasal, and temporal regions in

relation to the optic nerve (Elsheikh et al., 2010). .................................................................. 40

Figure 2.3: Illustration demonstrating how the eye cups were dissected .................................. 40

Figure 2.4: Overview of scleral tissue structure, including demonstration of melanocytes in

deeper sclera, from donor 6397L to determine integrity of tissue and basic structures

using H&E. .................................................................................................................................. 41

Figure 2.5: Bleaching optimisation results observed for donor 6397L (tissue taken from

lateral region). ............................................................................................................................. 42

Figure 3.1: IHC – SMA – performed on donor 6397L (sections taken from inferior region)....52

Figure 3.2: IHC – GABAA Alpha 1 – performed on donor 6397L (sections taken from inferior

region). ......................................................................................................................................... 53


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Figure 3.3: Shown above is the percentage of live scleral‐derived stromal cells (passage 3)...55

Figure A1: Animal models depicting the processes involved during induced‐hyperopia

myopic defocus (portrayed on the left) and induced‐myopia hyperopic defocus

(portrayed on the right) (Rymer and Wildsoet, 2005) ........................................................... 90

Figure A2: Some of the major muscles surrounding the eyeball; shown are the relative

positions of the insertions of the horizontal and vertical rectus muscles (Maza et al.,

2012).............................................................................................................................................. 92

Figure A3: The insertions of the superior oblique and inferior oblique muscles (Maza et al.,

2012).............................................................................................................................................. 92

Figure A4: Vascular supply to the globe, highlighting the relationships between the internal

carotid, ophthalmic, central retina, long posterior ciliary, short posterior ciliary, anterior

ciliary, and lacrimal arteries (Maza et al., 2012) ..................................................................... 93

Figure A5: Sites of perforation of the sclera by the long and short posterior ciliary arteries,

the short posterior ciliary veins, and the inferior and superior vortex veins (Maza et al.,

2012).............................................................................................................................................. 93

Figure A6: The relationships between the lacrimal, vortex, short posterior ciliary, inferior

ophthalmic, retinal, supraorbital, and superior ophthalmic veins (Maza et al., 2012) ..... 94

Figure A7: The relationships, shown as a transverse section of the globe, between the short

posterior ciliary, long posterior ciliary, and anterior ciliary arteries and short posterior

ciliary, anterior ciliary, and vortex veins (Maza et al., 2012) ................................................ 94


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ABBREVIATIONS, ACRONYMS, & SYMBOLS

α Alpha

Delta

Epsilon

Gamma

Pi

Theta

AGRF Australian Genome Research Facility

AL Axial Length

APC Allophycocyanin

BMC [3H]Bicuculline Methochloride

BP Base Pair

BSA Bovine Serum Albumin

CACA cis‐4‐Aminocrotonic Acid

CAMP cis‐2‐(aminomethyl)Cyclopropane‐1‐carboxylic Acid

cAMP Cyclic Adenosine Monophosphate

cDNA Complementary Deoxyribonucleic Acid

CNS Central Nervous System

DAB 3,3‐Diaminobenzidine

dNTP Deoxyribonucleotide Triphosphate

ECM Extracellular Matrix

EST Expressed Sequence Tag

FBS Fetal Bovine Serum

FC Flow Cytometry

FITC Fluorescein Isothiocyanate

GABA ‐Aminobutyric Acid

GABARAP GABAA Receptor‐Associated Protein

GABA‐T GABA Transaminase

GABAA GABAA Receptor α Subunit

GABRB GABAA Receptor β Subunit

GABRB3 GABAA Receptor β3 Subunit

GABRG GABAA Receptor Subunit GABRE GABAA Receptor Subunit GABRD GABAA Receptor Subunit GABRQ GABAA Receptor Subunit GABRP GABAA Receptor Subunit GABBR GABAB Receptor

GABRR GABAC Receptor Subunit GAD Glutamic Acid Decarboxylase

GAG Glycosaminoglycan

GAPDH Glyceraldehyde‐3‐Phosphate Dehydrogenase

I4AA Imidazole‐4‐Acetic Acid

IGF Insulin‐like Growth Factor

IHBI Institute of Health and Biomedical Innovation

IHC Immunohistochemistry

IL‐1β Interleukin‐1β


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IOP Intraocular Pressure

M1 – M5 Muscarinic Receptor Subtypes 1‐5

MMP Matrix Metalloproteinase

mRNA Messenger Ribonucleic Acid

NE Norepinephrine

NGF Nerve Growth Factor

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

PDGF Platelet‐Derived Growth Factor

PE Phycoerythrin

PG Proteoglycan

QEB Queensland Eye Bank

QEI Queensland Eye Institute

QUT Queensland University of Technology

RNA Ribonucleic Acid

RPE Retinal Pigment Epithelium

RT‐PCR Reverse‐Transcriptase PCR

SLRP Small Leucine‐Rich Proteoglycans

SMA Smooth Muscle Actin

TACA trans‐4‐Aminobut‐2‐enoic acid

TAMP trans‐2‐Aminomethylcyclopropanecarboxylic Acid

TGF Transforming Growth Factor

THIP 4,5,6,7,‐Tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol

TNF‐α Tumour Necrosis Factor‐α

TPMPA 1,2,5,6‐Tetrahydropyridine‐4‐yl)methylphosphinic Acid

VIP Vasoactive Intestinal Peptide


xiv

Statement of original authorship

The work contained in this thesis has not been previously submitted for a degree or diploma

in any other higher education institution. To the best of my knowledge and belief, the thesis

contains no material previously published or written by another person except where due

reference is made.

QUT Verified Signature

..................................................................................................................

Emily F. McDonald

February 28th 2014

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐

Structure of thesis

This Thesis has the following structure:

Chapter 1 – Literature Review

Chapter 2 – Materials and Methodology

Chapter 3 – Results

Chapter 4 – Discussion


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Acknowledgements

Katrina Schmid, thank you for teaching me how to be an independent researcher. I

appreciate your constructive and expert feedback on the drafts of this thesis.

Damien Harkin, I would like to thank you for your guidance throughout the histological

components of this project. Your passion for histology is inspiring. I agree – SMA is a rather

photogenic antibody.

Sally‐Anne Stephenson, I really appreciate your time, availability, and guidance throughout

the gene expression component of this project. Of course, I must mention my gratitude for

your muscles; without them, I would not have been able to extract RNA from the fibrous

scleral tissue. That was one of the best days in the lab for sure.

Neil Richardson, thank you for all the constructive discussions and the hours you have spent

in the lab guiding me through immunohistochemistry. Your easy‐going, encouraging

approach made the transition back into the world of science that much easier.

Laura Bray, I want to thank you for always being available – including at the lab bench.

Most notably, thank you for the hours spent on flow cytometry. On a personal note, your

friendship is truly invaluable and I would not be where I am without your support along the

way.

Tom Hogerheyde, thank you for teaching me the gene expression techniques. The

painstaking hours you put in were greatly appreciated.

Mark Woolf, without you pushing me I would not have taken on the project in the first place

and I am grateful. Thank you for your support.

To all my IHBI friends – you know who you are. My Masters experience would not have

been the same without each and every one of you. Thank you for your individual

contributions and I am looking forward to seeing you all enjoy success in your own fields.


xvi

Neil Tindale and Anne Roiko, I am forever indebted to both of you for the scientific

foundation and mentorship you provided me with during my years at USC.

On a personal note, I would like to thank my husband, Pete. You are my rock and none of

this would have been possible without you. I dedicate my thesis to you.

Emily F. McDonald


1

1 literature review

1.1 Introduction

Myopia, or short‐sightedness, is the most common refractive disorder (Young et al., 2007,

Tan et al., 2010, Young et al., 2003, Fredrick, 2002, Leo and Young, 2011) affecting

approximately 25% of the world population (Pan et al., 2012). The reasons behind the high

prevalence of myopia are currently obscure (Nickla and Wallman, 2010, Bloom et al., 2010,

Hammond et al., 2004, Brand et al., 2007) but its public health and economic impact is

considerable (Young et al., 2007, Baltussen et al., 2009, Kleinstein et al., 2003, Jones et al.,

2007, Rose et al., 2008b, Yang et al., 2009, Paluru et al., 2004). The impact of refractive errors

in Australia is significant (refer to Figure 1.1).

Figure 1.1: Leading causes of visual loss in Australia (Baird et al., 2010). Refractive error

correctable with spectacles is a significant contributing factor.

Myopia is predominantly caused by an elongated axial length (AL) of the globe (McBrien

and Gentle, 2003, Summers Rada et al., 2006). Less commonly, myopia is caused by the eyeʹs

optics; for example, a too powerful cornea or crystalline lens (Flitcroft, 2012). The sclera

must expand to allow for this increase in size and, ultimately, must grow, or remodel, for

myopia to develop (Young et al., 2004, Summers Rada and Hollaway, 2011, Barathi and

Beuerman, 2011, McBrien et al., 2006, Moring et al., 2007, Choo, 2003). Due to the location of

the retina and the sclera, the retinal signal must be transmitted to the sclera via the retinal

pigment epithelium (RPE) and choroid (Rymer and Wildsoet, 2005, Crewther, 2000). It is

generally considered that the retina is the primary controller of eye growth (Wallman and


2

Winawer, 2004). Evidence for this is the fact that when hemifield diffusers are used to form‐

deprive only half of the eye, only the half of the eye that was deprived elongates (Wallman et

al., 1987). Regional modifications to eye shape could not be produced via a signal sent from

the brain to the eyes.

The choroid and RPE have thus been implicated in eye growth control processes. It is

thought that the choroid might participate in refractive adjustment as a slow accommodative

mechanism: animal studies have demonstrated that the choroid is capable of increasing its

thickness in response to myopic defocus (Wallman et al., 1995) and thinning in response to

hyperopic defocus (Wildsoet and Wallman, 1995) The RPE is believed to play a critical role

in ocular growth regulation and the basis for this understanding is due to the fact that the

RPE separates the retina (the source of growth‐regulating signals) from the choroid and

retina and, accordingly, is involved in their growth regulation (Rymer and Wildsoet, 2005,

Crewther, 2000). Evidence suggests that RPE cells both synthesise and secrete many kinds of

cytokines (Tanihara et al., 1997). During negative lens treatment in chicks, insulin‐like

growth factor‐1 (IGF‐1) was observed to increase and IGF‐1 receptors were up‐regulated in

RPE, as well as in choroid and fibrous sclera (Penha et al., 2011). Chick RPE has been shown

to have β‐adrenoceptors, vasoactive intestinal peptide (VIP) receptors, α1‐adrenoceptors,

dopamine receptors, and muscarinic acetylcholine receptors (Rymer and Wildsoet, 2005).

It has been found that ‐aminobutyric acid (GABA), a retinal neurotransmitter, influences

eye growth and, accordingly, refractive development (Stone et al., 2003, Leung et al., 2005).

In the chick animal model of myopia, GABA antagonists have been shown to inhibit

experimental myopia (Chebib et al., 2009b) and have been proposed as having a ʺpromising

clinical role in the regulation of ocular growth including the treatment of myopia” (Leung et

al., 2005). Although the mechanism for this effect is unknown, it has been recently shown

that, in addition to the retina [human: (Davanger et al., 1991, Crooks and Kolb, 1992, Vardi

and Sterling, 1994, Gussin et al., 2011); chick: (Hering and Kröger, 1996, Albrecht and

Darlison, 1995)], GABA receptors are located in chick sclera (Cheng et al., 2011), chick cornea

(Cheng et al., 2012), and chick RPE (Cheng et al., In Press).

This project will determine if the three main GABA receptor sub‐types – GABAA, GABAB,

and GABAC [GABAC is actually a subset of GABAA (Bormann, 2000)] – are present or absent


3

in the human sclera. These receptor sub‐types were chosen as they constitute all known

GABA subtypes. Although previous studies have highlighted the influence of GABA on eye

growth and localisation of GABA receptors in chick sclera (Cheng et al., 2011, Leung et al.,

2005), no study has been undertaken involving human scleral tissue. A relatively recent

paper (Watson and Young, 2004) commented that it is surprising that the human sclera

remains such an under‐researched structure. The sclera had previously been thought to be

essentially inert (McBrien and Gentle, 2003) but it is now recognised that the sclera is a

dynamic tissue, capable of altering its composition and properties ‐ its rigidity and thickness

‐ in response to its visual environment (Summers Rada, 2006) and thus potentially playing a

role(s) in the onset and development of myopia (Watson and Young, 2004, Wang et al., 2011).

1.2 Brief Overview of the Eye

The eye (shown in Figure 1.2) is a nearly spherical organ consisting broadly of three layers:

1) the external layer formed by the cornea and sclera; 2) the intermediate layer divided into

two parts: the iris‐ciliary body anteriorly and the choroid posteriorly; 3) and the internal

layer consisting of the retina (Mitra et al., 2005). The eye is structurally limited by the cornea

and sclera (Ayoub, 2008). The iris controls, through automatic adjustment, the amount of

light entering the eye (Bogdanov, 2000). The ciliary body functions to produce components

of the aqueous humour (Fischer and Reh, 2003). The choroid is a thin, vascularised sheath

located between the sclera and the retina (Kiilgaard and Jensen, 2005). The retina, a

membrane containing an array of photoreceptor cells called rods and cones, converts light

energy into electrical signals (Bogdanov, 2000). The photoreceptors convert light energy to

electrical activity and transmit nerve impulses to the brain via the optic nerve (Bogdanov,

2000).


4

Figure 1.2: A pictorial representation of the human eye (Atchison and Smith, 2000).

1.3 Myopia: Definition, Epidemiology, & Possible Causes

Myopia (derived from the Greek words myein, meaning ‘to shut’ and ōps, meaning ‘eye’; i.e.,

to squint) is typically due to axial elongation of the vitreous chamber – ‘axial myopia’

(McBrien and Gentle, 2003, Summers Rada et al., 2006). ‘Axial elongation’ refers to

elongation of the eye in the axial direction; in myopia, the eye expands more axially than it

does equatorially (Atchison et al., 2004). The elongation process creates a mismatch between

the refractive power and length of the eye, the visual image of distant objects is formed in

front of the photoreceptor plane, and negative spectacle lenses are required to optically

correct the resultant image blur (Lundström et al., 2009, McBrien and Gentle, 2003,

Benavente‐Perez, 2006, Leo and Young, 2011, Metlapally and McBrien, 2008, Chen et al.,

2011). The refractive distribution of neonatal eyes is normal; over a period of approximately

six months, eyes emmetropise and, during this time, visual feedback is used to adjust

refraction to eliminate neonatal errors (Candy et al., 2009).

Myopia is considered to be a leading cause of visual impairment (Gilmartin, 2004, Fredrick,

2002, Majava et al., 2007). Severe cases of myopia, or high myopia, can lead to premature

cataracts, glaucoma, retinal detachment, and macular degeneration (Young, 2004, Saw et al.,

2005). Low myopia is observed around an AL of 24 mm, or 0 ‐ ‐6 dioptres, and high myopia

is observed around an AL of 30 mm, or greater than ‐6 dioptres (Meng et al., 2011). The

precise cause(s) or mechanism(s) triggering the axial elongation observed in myopia are still

unknown (Atchison et al., 2004, Shelton and Summers Rada, 2009, Tan et al., 2010, Wang et


5

al., 2011); myopia is regarded as a multifactorial, complex disorder (Chen et al., 2011). The

main conundrum concerns the fact that not all eyes develop myopia although being exposed

to comparable visual environments (Lundström et al., 2009).

Suggested causes and/or contributing factors include both genetic (refer to Baird et al., 2010

for a summary of myopia candidate genes) and environmental, such as education,

metabolism, and physical and outdoor activity (Chen et al., 2003, Yang et al., 2009, Fredrick,

2002, Paget et al., 2008, Hornbeak and Young, 2009, Jacobi and Pusch, 2010). For example,

myopia is more common in children who have myopic parents (Jones et al., 2007, Jones‐

Jordan et al., 2010, Liang et al., 2004) and the refractive errors of identical twins are similar

(Lyhne et al., 2001, Dirani et al., 2008). It is suggested that myopic children spend more time

indoors doing nearwork and less time outdoors than non‐myopic children (Khader et al.,

2006, Rose et al., 2008b, Rose et al., 2008a).

A recent genome‐wide association study (Kiefer et al., 2013) featuring 45,771 participants

identified 22 significant associations (20 of which were novel). The candidate genes were

identified as follows: DLX1, PABPCP2, PDE11A, PRSS56, ZBTB38, BMP3, KCNQ5, LAMA2,

SFRP1, TOX, TJP2, KCNMA1, RGR, LRRC4C, DLG2, RDH5, ZIC2, BMP4, GJD2, RASGRF1,

RBFOX1, and SHISA6. The most recent evidence is stronger for environmental causes

and/or contributing factors than for genetic (Lougheed, 2014, Goldschmidt and Jacobsen,

2014). It has also been suggested that myopia might develop either as a result of form‐

deprivation (Mathur et al., 2009), altered release of certain neurotransmitters and growth

factors (Yang et al., 2009, Raviola and Wiesel, 2007), diet (Charman, 2011, Mutti and Marks,

2011, Cordain et al., 2002), or relative peripheral hyperopia, where the abnormal axial

growth of the eye is caused by the peripheral image lying behind the retina (Mathur et al.,

2009, Mutti et al., 2007).

1.4 The Proposed Myopia Signalling Cascade: Where the Sclera is thought to fit in

Due to the fact that the retina directly processes visual information, it has also been

suggested that it is the most likely candidate to generate the primary signal for myopia

development (Wallman, 1993). This potential process, initiated by a visual stimulus and

resulting in a cascade of events through the different layers of the eye, is depicted in Figure

1.3 shown below (Young, 2010). As shown in Figure 1.3, when the AL of the eye is shorter


6

than the focal plane, hyperopic defocus (1) occurs on the retina unless fully cleared by

accommodation (9). Hyperopic defocus reduces the amplitude of responses of retinal

neurons (2); in turn, the communication of signals (3) through the RPE and (4) choroid is

altered. The resulting communication to the sclera produces (5) altered gene expression in

fibroblasts. Eventually, remodelling of the scleral extracellular matrix (ECM) occurs (6),

increasing the creep rate of the sclera (7), which increases the axial elongation rate of the eye

(8). The amount of defocus is reduced as the axial elongation moves the retina closer to the

focal plane. Responses increase with reduced retinal defocus and, in turn, alter

communication to the sclera. Finally, scleral remodelling is altered such that both the creep

rate is decreased and axial elongation is slowed. The following section addresses the

functional anatomy and potential roles of the retina, RPE, choroid, and sclera in the

proposed visually‐driven signalling pathway. As the focus of this project is on the sclera, it

is particularly important to briefly highlight that, as the sclera forms the structural outer coat

of the eye, in conjunction with the choroid, it controls the location of the retina (Young,

2010).

Figure 1.3: An emmetropisation model depicting the visual signalling cascade (Young, 2010).

The cascade of events involves the retina, RPE, choroid, and sclera.

1.4.1 Neural Retina: Generates Primary Signal for Myopia Development?

The neural retina (refer to Figure 1.4 for a pictorial representation of the retinal layers),

approximately 100‐200 μm thick, is responsible for processing all incoming visual signals

(Massey, 2005). The retinal layer, a thin sheet at the back of the eye responsible for

converting light associated with visual image into action potentials that are sent to the brain,

contains six discrete populations of cells: photoreceptors (rod and cones), horizontal cells

(lateral interneurons), bipolar cells (vertical connections), amacrine cells (lateral

interneurons), ganglion cells (output neurons), and interplexiform cells (Massey, 2005).

The human retina contains a number of neurotransmitters and neuropeptides, such as

dopamine, glycine, substance P, and somatostatin (Tornqvist and Ehinger, 1988); the


7

prevailing retinal neurotransmitter molecules implicated in influencing eye growth

regulation are dopamine (Schaeffel et al., 1995, Wallman, 1993), muscarinic acetylcholine (Lin

et al., 2009, McBrien et al., 2001b), glucagon (Fischer et al., 1999), and VIP (Tornqvist and

Ehinger, 1988, Rymer and Wildsoet, 2005). Refer to Flitcroft (2012) for a recent review

summarising current knowledge on the role of the retina in the control of eye growth. The

next innermost layer of the eye is the RPE. The following section outlines the functional

anatomy of the RPE and its potential role in the local signalling cascade mediating myopia.

Figure 1.4: The different layers of the retina and the cells associated with each of these layers

(Yang, 2004). GABAergic neurons are shown in blue. GABA is typically involved in the

lateral pathways involving horizontal and amacrine cells.

There is substantial evidence from animal studies to support the role of the retina in the

regulation of ocular growth. For example, if the optic nerve is severed or the action

potentials in it blocked, form‐deprivation myopia is unaffected (Norton et al., 1994, Troilo et

al., 1987, Wildsoet and Pettigrew, 1988). In addition, lens compensation occurs, although

with some differences (Wildsoet, 2003, Wildsoet and Wallman, 1995). It has been observed

in studies that use diffusers or negative lenses that cover only half of the retina, only the

covered half of the eye exhibits myopia; in contrast, if positive lenses are used to cover half


8

of the retina, only the covered half exhibits inhibited eye growth (Diether and Schaeffel,

1997, Hodos and Kuenzel, 1984, Wallman et al., 1987).

1.4.2 Retinal Pigment Epithelium: Signal Relay in the Control of Eye Growth?

The RPE, classically described as a polarised epithelial monolayer of cells, is sandwiched

between the retina and the choroid; due to its location, the RPE is thought to play a crucial

role in relaying signals between the retina and choroid (Rymer and Wildsoet, 2005,

Crewther, 2000). Refer to Table 1.1 for a list of functions of the RPE.


9

Table 1.1: Known functions of the RPE as compiled by Strauss in 2005 (Strauss, 2005). The

RPE is responsible for a diverse array of functions, including light absorption, transportation,

nutrient absorption, phagocytosis, and secretion.

Functions of RPE Reference(s)

Absorbs the light energy focused by the lens on the retina (Bok, 1993, Boulton and

Dayhaw‐Barker, 2001)

Transports ions, water, and metabolic end products from the

subretinal space to the blood

(Dornonville de la Cour,

1993, Hamann, 2002,

Marmor, 1999, Miller and

Edelman, 1990, Steinberg,

1985, Crewther, 2000)

Absorbs nutrients such as glucose, retinol, and fatty acids from the

blood and delivers these nutrients to photoreceptors

(Strauss, 2005)

Enables exchange of retinal between the photoreceptors and the RPE;

this is important as photoreceptors are unable to reisomerise all‐

trans‐retinal, formed after photon absorption, back into 11‐cis‐

retinal. Therefore, retinal is transported to the RPE, reisomerised to

11‐cis‐retinal and transported back to the photoreceptors: this

process is known as the ‘visual cycle of retinal’.

(Strauss, 2005, Baehr et al.,

2003, Besch et al., 2003,

Thompson and Gal, 2003,

Booij et al., 2010)

Stabilises ion composition in the subretinal space, which is essential

for the maintenance of photoreceptor excitability

(Dornonville de la Cour,

1993, Steinberg, 1985,

Steinberg et al., 1983, Booij et

al., 2010)

Phagocytosis of shed photoreceptor outer segments; the outer

segments of the photoreceptors are digested, and essential

substances, such as retinal, are recycled and returned to the

photoreceptors to rebuild light‐sensitive outer segments from the

base of the photoreceptors.

(Bok, 1993, Finnemann,

2003, Gal et al., 2000,

Strauss, 2005, Strauss et al.,

1998, Wistow et al., 2002,

Booij et al., 2010)

Secretes a variety of growth factors helping to maintain the structural

integrity of choriocapillaris endothelium and photoreceptors

(Strauss, 2005)

Plays an important role in the establishing the immune privilege of the

eye by secreting immunosuppressive factors

(Ishida et al., 2003, Streilein

et al., 2002)

The RPE is critical to communication occurring between the retina and the rest of the eye.

Due to its central location within the outer layers of the eye, it is thought that the RPE may

act as a signal relay in the control of eye growth (Crewther, 2000). In their review paper,

Rymer & Wildsoet (2005) suggested that, aside from the presence of ion transporters and

signal receptors, the RPE, lying between the signal source (retina) and its target (the sclera

and choroid) may play a critical role based on location alone. In addition, Rymer & Wildsoet

(2005) highlighted three retinal neurotransmitters, dopamine, VIP, and glucagon, that have

been linked to eye growth regulation. As the RPE secretes growth factors into the choroid

targeting both the choroid and sclera, it is thought that the concentration and activity of

these growth factors might be modulated by fluid transport across the RPE (Crewther, 2000,

Rymer and Wildsoet, 2005). The next section addresses the functional anatomy and potential

role of the choroid in the myopia signalling cascade.


10

1.4.3 Choroid: Responsible for Fluid Regulation

The term choroid is derived from the Greek words for “membrane” and “form”; the choroid

(the middle tunic of the eye) is a vascularised and pigmented tissue attached to the sclera by

strands of connective tissue (Guyer et al., 2005). The choroidal circulation demonstrates one

of the highest rates of blood flow – millilitres/minute/gram – in the human body (Alm and

Bill, 1973, Alm and Bill, 1987, Torczynski, 1987); more than 70% of all the blood in the globe

at any one time can be found in the choriocapillaris (Parver et al., 1980, Rohen, 1964), one of

the layers of the choroid. From the most superficial to the deepest, the four layers of the

choroid are termed the suprachoroid or suprachoroidea: a transitional zone between the

choroid and sclera containing elements of both, stroma: extravascular tissue, choriocapillaris:

a highly anastomosed network of capillaries, and Bruch’s membrane: a complex 5‐laminar

structure (Guyer et al., 2005, Nickla and Wallman, 2010). In terms of composition, the

choroid is comprised of blood vessels, melanocytes, fibroblasts, resident immunocompetent

cells and supporting collagenous and elastic connective tissue (Nickla and Wallman, 2010).

In humans, the choroid is approximately 200 μm in width at birth and decreases to

approximately 80 μm by the age of 90 (Ramrattan et al., 1994). Choroidal thickness is

diurnally modulated: the choroid is thickest at about midnight and thinnest at noon

(Papastergiou et al., 1998, Nickla et al., 1998). It is believed that this process is driven by a

circadian oscillator as the rhythm free‐runs in constant darkness (Nickla, 2006).

The main functions of the choroid are three‐fold: 1) thermoregulation via heat dissipation, 2)

nourishment of the RPE and retina up to the outer aspect of the inner nuclear layer, and 3)

producing the visible pigmentation of the fundus (Guyer et al., 2005). In addition, the

choroid secretes growth factors, adjusts the position of the retina by changes in choroidal

thickness, and plays an important role in the drainage of the aqueous humour from the

anterior chamber, via the uveoscleral pathway (Nickla and Wallman, 2010) [approximately

35% of the drainage in humans (Alm and Nilsson, 2009)]. A recent paper (Summers, 2013)

outlined the role of the choroid in ocular growth and the development of refractive errors as

follows: 1) the choroid can act on neighbouring tissues through growth factors, 2) choroidal

gene expression and thickness can be influenced by visual stimuli, 3) the choroid, in

response to visual stimuli, secretes scleral growth factors, and 4) choroidal RALDH2 activity

and retinoic acid synthesis have been linked to modulation of scleral remodelling.


11

1.4.4 Sclera: Responsible for Controlling Eye Dimensions

Finally, the outermost layer is the sclera. As the sclera is thought to control the dimensions

of the eye, it is important to assess the potential role of the sclera in the visually‐driven

signalling pathway. Essentially, the sclera controls the location of the retina as it forms the

structural outer layer of the eye (Young, 2010). In the context of the myopic eye, the

resultant thinning of the sclera during elongation is attributed to tissue remodelling and not

simply passive stretching (Choo, 2003). The sclera will be covered in greater detail –

including gross structure, function and biochemistry – in Section 1.6.

1.5 ‐Aminobutyric Acid

Neurotransmitters can be divided into two sub‐groups: inhibitory and excitatory (Wilson

and Cowan, 1972). GABA (the chemical structure shown in Figure 1.5) is an important

inhibitory neurotransmitter present in the central nervous system (CNS), which includes the

retina (Krogsgaard‐Larsen et al., 1997, Chebib et al., 2009b, Enz and Cutting, 1999, Barnard et

al., 1998). GABA was first discovered over 60 years ago in the mammalian CNS (Roberts and

Frankel, 1950). The neurotransmitter is most highly concentrated in the substantia nigra,

basal ganglia, hypothalamus, the periaqueductal grey matter, and the hippocampus (van der

Zeyden et al., 2008). In the mammalian brain, GABA‐releasing synapses are the principle

source of inhibition and glutamate‐releasing synapses are the principle excitatory

transmitters (Ben‐Ari, 2002); GABA and glutamate only differ by a single carboxyl group

(Waagepetersen et al., 2007). Approximately 90% of all synaptic neurotransmission in the

CNS is mediated by GABA and glutamate (Schousboe and Waagepetersen, 2003,

Waagepetersen and Schousboe, 2009) and at least one‐third of all CNS neurons utilise GABA

as their primary neurotransmitter (Vithlani et al., 2011). GABAergic transmission requires

both a synthesising enzyme – glutamic acid decarboxylase, or GAD – and a degradative

enzyme – GABA transaminase, otherwise known as GABA‐T (Waagepetersen et al., 2007,

Yazulla, 2010).

Figure 1.5: Chemical structure of ‐aminobutyric acid (Ng et al., 2011).


12

The role and significance of GABA receptors in non‐neuronal human tissue is obscure.

Recent research using tissue obtained from mice found GABAA receptor protein in the

following non‐neuronal sites: renal medulla, cortex, heart, left ventricle, aorta, and pancreas

(Tyagi et al., 2007). GABAA receptor protein has also been found to be present in human,

rabbit, and rat kidney tissue (Sarang et al., 2001). Future studies determining the role and

significance of GABA receptors in non‐neuronal tissues, including sclera, would help in

piecing together the big picture of myopia and would shed some light on potential

pharmaceutical intervention.

1.5.1 ‐Aminobutyric Acid Receptors – GABAA, GABAB, and GABAC

GABA is a highly flexible molecule; it can attain many low‐energy conformations that bind

to three pharmacologically and physiologically distinct GABA receptors: GABAA, GABAB,

and GABAC (Figure 1.6, Figure 1.7, & Table 1.2). GABAC is, more recently, known as

GABAA0r (Barnard et al., 1998, Leung et al., 2005, Bormann, 2000); the change in

nomenclature classifies the ‐containing GABA receptors as a specialised set of GABAA

receptors that are insensitive to both bicuculline and benzodiazepines (Bormann, 2000).

Generally, GABAA and GABAB receptors are defined by their respective sensitivities to

bicuculline and baclofen; GABAC receptors are a pharmacologically distinct group – they do

not respond to either drug (Bowery, 1989, Johnston, 1996, Bormann and Feigenspan, 1995,

Cherubini and Strata, 1997, Bormann, 2000); see Figure 1.8.


13

A)

B)

C)

Figure 1.6: Structure of the GABAA receptor. [Figure (A) taken from (Enna, 2007); Figures (B)

and (C) taken from (Akk et al., 2007).]

(A) Pentameric structure of GABAA receptor showing five representative subunits – in this

case, 2x α subunits, 2x β subunits, and 1x 2 subunit (Enna, 2007). Also shown are unique

binding sites for each subunit. The binding sites for GABA are sandwiched between the α

and β subunits. Ionotropic GABAA receptors are fast‐acting, ligand‐gated chloride channels


14

(Sieghart, 2006); GABA receptor activation causes an influx of chloride ions (Waagepetersen

et al., 2007). The membrane topography of GABAC receptors is assumed to be very similar to

that of GABAA (Bormann, 2000). However, GABAC receptors are composed exclusively of (1‐3) subunits (Bormann, 2000). (The two P symbols at the bottom of the diagram represent

protein kinase.)

(B) A single subunit of the GABAA receptor (Akk et al., 2007). This image is focusing on the

topography of the subunits; M1‐M4 represents individual transmembrane domains. The

transmembrane domain coloured grey – M2 – forms an important part of the chloride

channel (Akk et al., 2007).

(C) Top‐down view of the pentameric structure of the GABAA receptor (Akk et al., 2007).

Figure 1.7: Structure of the GABAB receptor (Enna, 2007).

Metabotropic GABAB receptors produce slow and prolonged inhibitory responses; GABAB

receptors are coupled indirectly via proteins to either calcium or potassium channels (Bettler

and Tiao, 2006). Postsynaptically, stimulation of GABAB receptors causes an efflux of

potassium ions (Waagepetersen et al., 2007); presynaptically, GABAB receptors regulate

calcium channels and thereby cause inhibition of transmitter release (Olsen and Betz, 2006).

It is important to note that the GABAB receptor is an obligate heterodimer: the receptor is

only functional when both the GABAB R1 isoform and the GABAB R2 isoform are co‐

expressed in the same cell (Pierce et al., 2002).


15

a)

b)

Figure 1.8: Pharmacological differences between GABAA and GABAC receptors (Gottesmann,

2002, Bormann, 2000).

(A) The GABAA receptor has binding sites for barbiturates, benzodiazepines, and

neurosteroids and GABA responses are blocked by bicuculline (competitively) and by

picrotoxin (non‐competitively) (Gottesmann, 2002, Bormann, 2000).

(B) The GABAC receptor is activated by CACA, antagonised by TPMPA, and blocked by

picrotoxinin (Gottesmann, 2002, Bormann, 2000).


16

Table 1.2: Characteristics of the three GABA receptor types: GABAA, GABAB, and GABAC.

GABAA Receptor GABAB Receptor GABAC (or GABAA0R)

Receptor

Reference(s)

Category Ligand‐gated ion channel G‐protein coupled

receptor (to either calcium

or potassium channels)

Ligand‐gated ion channel (Emberger et al., 2000, Enz and Cutting, 1999,

Johnston, 1996, Ben‐Ari et al., 2007, Kumar et

al., 2008, Vithlani et al., 2011)

Subunits α1‐6, β1‐3, 1‐3, , , , & GBR1 & GBR2 1‐3 (Bormann, 2000, Krogsgaard‐Larsen et al.,

1997, Fritschy et al., 2004, Emberger et al.,

2000, Enz and Cutting, 1999, Schousboe and

Waagepetersen, 2008, Stone et al., 2003, Ben‐

Ari et al., 2007, MacDonald et al., 2007,

Vithlani et al., 2011)

Agonists I4AA

Isoguvacine

Isonipecotic acid

Muscimol

TACA

TAMP (weak)

THIP

Baclofen CACA

CAMP

Muscimol (partial)

TACA

TAMP

(Bormann, 2000, Krogsgaard‐Larsen et al.,

1997, Schousboe and Waagepetersen, 2008)

Antagonists BMC

Gabazine

Picrotoxin

Phaclofen

Saclofen

I4AA

Isoguvacine (weak)

Picrotoxin

THIP (weak)

TPMPA

(Bormann, 2000, Krogsgaard‐Larsen et al.,

1997, Enz and Cutting, 1999)

Desensitisation Strong Variable Weak (Bormann, 2000, Mutneja et al., 2005)

Modulator(s) Barbiturates

Benzodiazepines

Ethanol

Neurosteroids

Triazolopyridazines

‐‐‐ Zinc? (Bormann, 2000, Emberger et al., 2000,

Kaneda et al., 2000)

BMC – [3H]bicuculline methochloride; CACA – cis‐4‐aminocrotonic acid; CAMP – cis‐2‐(aminomethyl)cyclopropane‐1‐carboxylic acid; I4AA – imidazole‐4‐acetic acid; TACA –

trans‐4‐aminobut‐2‐enoic acid; TAMP – trans‐2‐aminomethylcyclopropanecarboxylic acid; THIP – 4,5,6,7,‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol; TPMPA – and (1,2,5,6‐

tetrahydropyridine‐4‐yl)methylphosphinic acid.


17

1.5.2 ‐Aminobutyric Acid as a Piece in the Myopia Puzzle: GABA in Eye Growth

GABA antagonists have been found to influence ocular growth in chick models of myopia

(Stone et al., 2003, Leung et al., 2005) and, in association with inhibition of myopia

development in chicks, facilitate learning and memory in mice (Chebib et al., 2009b). One

key study (Stone et al., 2003) demonstrated that GABA plays a modulatory role during eye

growth and refractive development. Refer to Table 1.3 for a summary of papers addressing

the use of GABA in animal models.

Table 1.3: Summaries of notable papers utilising GABA in animal studies. GABA

antagonists were found to inhibit both form‐deprivation myopia and normal eye

development.

Reference(s) Background Finding

(Stone et al.,

2003)

White leghorn chicks received daily intravitreal

injections of agonists or antagonists to GABAA

and GABAC Rho receptor subtypes. Results

following the injection period were determined

using refractometry, ultrasound, and callipers.

Antagonists to GABAA and

GABAC Rho receptor

subtypes inhibited both

form‐deprivation myopia

and the development of eyes

with normal visual input.

(Leung et al.,

2005)

Gallus domesticus chicks received regular (days 4, 7

& 10) intravitreal injections of two antagonists –

bicuculline and TPMPA – to GABAA and

GABAC Rho receptor subtypes. Measurements

following the trial period were taken using

ultrasound, streak retinoscopy, and micrometer

screw gauges.

Two antagonists – bicuculline

and TPMPA – to GABAA

and GABAC Rho receptor

subtypes inhibited both

form‐deprivation myopia

and normal eye

development.

(Chebib et al.,

2009b)

Red‐Rhode Island White cross cockerels were fitted

monocularly with a ‐15D spectacle lens and

received daily intravitreal injections of cis‐3‐

ACPBPA or trans‐3‐ACPBPA, GABAC

antagonists. Measurements were taken using

streak retinoscopy and A‐scan ultrasonography.

Mice received intra‐peritoneal injections of cis‐3‐

ACPBPA or trans‐3‐ACPBPA and, following the

injection period, were subjected to the Morris

Water Maze to determine potential effects of the

above‐mentioned drugs on learning and

memory capability.

Antagonists to GABAC – cis‐3‐

ACPBPA and trans‐3‐

ACPBPA – were found to

both inhibit myopia

development in chicks and

facilitate learning and

memory in mice.

1.5.3 ‐Aminobutyric Acid within the Human Eye

The human retina contains all three GABA receptor subtypes (Qian and Ripps, 2009). The

majority of the mammalian retinal cells found to express GABA are the amacrine cells

(Nguyen‐Legros et al., 1997, Davanger et al., 1991, Crooks and Kolb, 1992). GABAC is

expressed predominantly in the retina on bipolar cells of every subtype (Qian and Ripps,

2009). In the retina, GABA shares functional pathways with dopamine and acetylcholine,


18

neurotransmitters also implicated in refractive development (Stone et al., 2003). Refer to

Table 1.4 for general and specific locations of GABA receptors in the eye (both human and

animal due to somewhat limited human data).

A study was undertaken by Young in 2003 using human scleral tissue from nine donors with

non‐myopic refractive history (Young et al., 2003). A cDNA (complementary

deoxyribonucleic acid) library was constructed from the scleral ribonucleic acid (RNA) and a

total of 640 expressed sequence tags (ESTs) were produced. The only neuronal‐specific EST

yielded was for GABAA receptor‐associated protein (gene name: GABARAP). [It must be

noted that this study was by no means comprehensive. The authors acknowledged this

point based on the fact that genes such as collagen type 1 and elastin, known constituents of

the sclera, were not identified in their library screening procedure. The authors estimated

that the genes that were identified in the study represented only 1‐2% of the total number of

mRNA (messenger ribonucleic acid) species potentially expressed by scleral fibroblasts

(Young et al., 2003).]

It has been postulated that, in the normal eye, GABA may be involved in determining the

shape of the normal developing eye (Leung et al., 2005). One paper (Leung et al., 2005) has

suggested, based on animal studies undertaken in chicks, that GABA antagonists may have a

direct effect on ocular growth; accordingly, GABA antagonists have been recommended as

having “a promising clinical role in the regulation of ocular growth including the treatment

of myopia” (Leung et al., 2005). Leung et al. (2005) found that the GABA antagonists,

bicuculline and TPMPA, reduced form‐deprived ocular growth, as well as normal growth.

However, it must be noted that bicuculline alone, at a concentration of 10 mg / mL, caused

hypermetropia of the control eyes.

1.5.4 ‐Aminobutyric Acid in Animal Models

A recent study (Cheng et al., 2011) found the GABAC receptor subtype, rho1, present in chick

sclera (the presence or absence of the other two main receptor subtypes was not

investigated). Another recent study (Cheng et al., 2012) found the GABAA Alpha 1 and

GABAC Rho 1 receptor subtypes present in chick cornea; GABAB was not found to be

present. A study currently in press (Cheng et al., In Press) reported expression of GABAA

Alpha 1, GABAB R2, and GABAC Rho 1 receptor subtypes in chick RPE. Although GABA


19

receptors have been found in chick sclera (and cornea and RPE), it is unknown whether

GABA receptors are present or absent in human sclera. For background information into

animal studies – species employed, techniques used, and rationale for different approaches,

refer to Appendix 1.


20

Table 1.4: Ocular tissues – human and animal – found to have GABA receptors present.

GABA‐stained retinal cells include amacrine and ganglion cells.

Ocular Tissue GABA Present in: Finding Reference(s)

Human Retina The total number of GABA‐stained cells

(strongly or moderately stained) was

estimated to be between 26 and 40%. The

majority of cells that demonstrated GABA

immunoreactivity were interpreted as

amacrine cells, terminating in the inner

plexiform layer (as none of the stained cells

displayed extensions in an outward

direction). Furthermore, a subpopulation

was observed to be immunoreactive in the

ganglion cell layer (these cells were

assumed to be displaced amacrine cells).

Cells thought to represent sectioned

interplexiform cell fibres and terminals

present in the inner nuclear and outer

plexiform layers appeared to demonstrate

GABA immunoreactivity. There appeared

to be some GABA‐stained ganglion cells

but no horizontal, bipolar, or Müller cells

stained for GABA.

(Davanger et

al., 1991)

Human Retina The study focused on the central retina.

Approximately 40% of the amacrine cells

present in the inner nuclear layer were

found to be GABA immunoreactive.

Furthermore, a few amacrine cells in the

inner plexiform layer and ganglion cell

layer also demonstrated GABA

immunoreactivity. There appeared to be

faint staining of photoreceptors and cone

pedicles in the outer plexiform layer.

(Bipolar, horizontal, and most ganglion

cells did not demonstrate

immunoreactivity.)

(Crooks and

Kolb, 1992)

Human Retina Staining for GABAA Alpha 1 subunit was

found to be intense in both the inner and

outer plexiform layers. Immunoreactivity

for the above antibody was also present in

many amacrine and ganglion cell somas in

the inner nuclear layer and ganglion cell

layer. The same cells also stained for

GABAA Beta 2 & 3 but less intensely. The

presence of the GABAA receptors on the

bipolar dendritic tips may mean that the

role of the GABA receptors are to mediate

the receptive field surrounding both off and

on bipolar cells. The presence of the

GABAA receptors on the bipolar axon

terminals suggest that much of the

inhibition feeding back from GABAergic

amacrine to bipolar cells is GABAA‐

(Vardi and

Sterling, 1994)


21

mediated.

Vertebrate Retina

Although all three GABA receptor subtypes

were found to be present in the vertebrate

retina, the retina was found to be

particularly enriched with GABAC. Of

note, it was mentioned that “GABA‐

mediated inhibition may play important

roles in modulating visual information as it

flows from photoreceptors to the brain”.

(Lukasiewicz

and Shields,

1998)

Human Retina GABAC Rho 1 immunoreactivity was primarily

observed in the inner plexiform layer.

(Gussin et al.,

2011)

Chick Sclera

The GABAC Rho 1 receptor subunit was found

to be present in fibroblasts and

chondrocytes of both the fibrous and

cartilaginous layers of chick sclera.

(Cheng et al.,

2011)

Chick Cornea

Chick corneal tissue was found to be positive

for both GABAA Alpha 1 and GABAC Rho 1

receptor subunits and was negative for

GABAB. GABA may play a role in corneal

transparency by way of fluid transportation

in the corneal epithelium. The GABA

receptors may be involved in regulating

corneal sensitivity or other neural functions

in the corneal epithelium.

(Cheng et al.,

2012)

1.5.5 ‐Aminobutyric Acid in Fibroblasts of other Tissues

The functional impact of GABA on fibroblast activity, in particular, has been documented in

a few relatively recent studies. For example, GABA stimulates the synthesis of hyaluronic

acid and enhances the survival rate of the dermal fibroblasts obtained from skin samples (Ito

et al., 2007). Less recently (Göhlich et al., 1984), it was demonstrated that the rate of GABA

synthesis in skin fibroblasts from Huntington’s disease patients can be around 3 times higher

than in healthy controls. A study on fibroblasts obtained from human buccal mucosa found

that GABA stimulated collagen synthesis and proliferation (Scutt et al., 1987). From a gene

expression perspective, several genes encoding cellular receptors, including GABAA, have

been noted in genes induced in skin and lung fibroblasts (Gu and Iyer, 2006). mRNA

expression for the GABAA receptor β3 subunit, GABRB3, has been found to be up‐regulated

in human cleft lip and/or palate fibroblasts (Baroni et al., 2006). The cell proliferation activity

of GABA was studied using the mouse fibroblast cell line, NIH3T3 (Han et al., 2007). It was

found that GABA treatment significantly promoted cell proliferation and significantly

suppressed the mRNA expression of inflammatory mediators, such as IL‐1β (interleukin‐1β)

and TNF‐α(tumour necrosis factor‐α) (Han et al., 2007). From the above studies, it is clear

that GABA can potentially play a functional role in fibroblast activity.


22

1.5.6 Pharmacological Factors Affecting Eye Growth in Animal Models

A recent review into pharmaceutical interventions for myopia control (Ganesan and

Wildsoet, 2010) addressed the use of GABA and cited three studies; two studies involved

chicks and one involved monkeys. The chick studies – one utilising form‐deprivation

myopia and the original study of its kind (Stone et al., 2003) and the other one lens‐induced

myopia (Chebib et al., 2009a) – both reported the same outcome: anti‐myopia effects for a

range of GABA antagonists. More specifically, GABAC‐selective antagonists proved to be

more potent than either GABAA‐ or GABAB‐selective antagonists. Both of these studies are

in agreement with another recent study (Chebib et al., 2009b), which also found GABA

antagonists – cis‐ and trans‐3‐ACPBPA – had weak effects (presence of the antagonists

resulted in a reduced cellular GABA response) at GABAA and GABAB receptors but potent

antagonist effects at GABAC receptors. The study involving monkeys found retinal GABAC

receptors appeared to be involved in modulation by GABA of a retinoic acid pathway (Song

et al., 2005). The review concludes the section on GABA with this: “However, there has been

no follow‐up to this study [referring to: (Song et al., 2005)] and no relevant studies of other

mammalian models” (Ganesan and Wildsoet, 2010).

1.6 Human Sclera

The sclera, or ‘skeleton’ of the eye (Trier, 2005), is a dynamic tissue; it is capable of

responding to changes, modulated by visually guided signals originating from the retina, in

the visual environment by altering ocular size and refraction (Summers Rada et al., 2006).

The biochemical and biomechanical properties of the sclera determine, to a large degree, the

shape and size of the globe and thus the refractive state; scleral physiology must alter to

allow the eye to expand during refractive development (Summers Rada et al., 2006,

Benavente‐Perez, 2006, Jobling et al., 2009, Young et al., 2003, Yang et al., 2009). Accordingly,

myopia development presents, not only as a rapid increase in eye size, but is also associated

with a loss of scleral tissue weight (Yang et al., 2009) and scleral thinning (Morgan, 2003).

However, it has not been fully elucidated what initiates and regulates the scleral remodelling

that occurs during myopia onset and development (McBrien et al., 2006). For relevant

background information, the following sections outline the different constituents and

biochemical properties of the sclera.


23

1.6.1 Gross Structure & Function of Sclera

The sclera (derived from the Greek word skleros, meaning ‘hard’), together with the cornea, is

derived from both the neural crest and mesoderm and forms the outermost covering of the

eye (Summers Rada et al., 2006, Kashiwagi et al., 2010, Young et al., 2003, Seko et al., 2008,

Fullwood et al., 2011). It is commonly known as the white of the eye and forms an almost

complete sphere between 22 mm (Tsai et al., 2010) and 24 mm diameter (Watson and Young,

2004). The sclera is a dense, fibrous, viscoelastic connective tissue (Summers Rada et al.,

2006, Rada et al., 1997, Yang et al., 2009), accounts for approximately 85% of the total ocular

surface (McBrien and Gentle, 2003, Watson and Young, 2004), and extends from the cornea

to the optic nerve (Elsheikh et al., 2010). The sclera is composed of three layers: (external

layer to inner aspect) the episclera, scleral stroma proper, and the lamina fusca (McCluskey,

2001, Watson and Young, 2004).

Scleral connective tissue contains matrix‐secreting fibroblasts embedded in a matrix of

irregular and interwoven collagen fibres in close association with various proteoglycans, or

PGs, and glycoproteins (Rada et al., 1997, Summers Rada et al., 2006, Young et al., 2003,

McCluskey, 2001, McBrien et al., 2006). The underlying structure, composition, and

arrangement of collagen fibres in the sclera presents very differently to collagen found in the

cornea; its presentation is more comparable to collagen found in the skin (Yang et al., 2009,

Summers Rada et al., 2006). Due to the irregularity of its collagen fibres and its relative

hydration, the sclera is characteristically opaque and yet brilliantly white (McBrien and

Gentle, 2003, Elsheikh et al., 2010, Meek and Fullwood, 2001, Kashiwagi et al., 2010, Tsai et

al., 2010, Fullwood et al., 2011). The opacity of the sclera ensures that internal light scattering

does not affect the retinal image (Watson and Young, 2004, Seko et al., 2008). The sclera is

avascular; it obtains its nutrients via the choroid and vascular plexi in Tenon’s capsule – a

hypocellular layer of radially‐arranged, compact collagen bundles running parallel to the

scleral surface – and episclera (Watson and Young, 2004).

The main functions of the sclera are essentially three‐fold: 1) the sclera is rigid, but not

inflexible, in order to offer resistance to the effects of intraocular pressure, or IOP, and

external trauma (Summers Rada et al., 2006, McBrien and Gentle, 2003, Elsheikh et al., 2010,

Rada et al., 1997, McCluskey, 2001, Tsai et al., 2010, Watson and Young, 2004); 2) the sclera is

tough and impenetrable – yet allowing vascular and neural access – in order to provide


24

protection to ocular components (McBrien and Gentle, 2003, Summers Rada et al., 2006,

Elsheikh et al., 2010, McCluskey, 2001, Tsai et al., 2010, Trier, 2005, Watson and Young, 2004);

and 3) the sclera is capable of rapid remodelling whilst maintaining shape and dimensional

parameters by providing a stable base for contractions of the ciliary muscle (McBrien and

Gentle, 2003) and enabling attachment for other extraocular muscle insertions (Elsheikh et

al., 2010, McCluskey, 2001).

1.6.2 Composition of the Sclera

The major biochemical components of the sclera include collagen, elastin, glycoproteins, and

proteoglycans (refer to Table 1.5). This structure is responsible for maintaining the rigidity,

strength, and elasticity that is characteristic of the sclera (Summers Rada et al., 2006). The

sclera essentially consists of connective tissue comprised of the ECM and ECM‐secreting

fibroblasts (Summers Rada et al., 2006) in close association with PGs and glycoproteins

(Young et al., 2004); refer to Figure 1.9. Biochemical changes in the scleral framework and in

the molecules required for synthesis and degradation processes can lead to significant

changes in scleral biomechanical properties; accordingly, associated biochemical and

biomechanical changes have been observed in scleral shape, ocular size, and, eventually, the

refractive state of the eye (Young et al., 2004, McBrien et al., 2009). Current knowledge

addressing specific biochemical and biomechanical changes during myopia onset and

development are described at the end of each section below, if applicable.

Figure 1.9: Model illustrating the likely molecular structure – based on current knowledge –

of the ECM of the mammalian sclera (McBrien and Gentle, 2003). Major biochemical

components include collagen, elastin, glyproteins, and proteoglycans.


25

Table 1.5: Main biochemical constituents present in human sclera.

Constituent Reference(s)

Fibrous Connective Tissue (Summers Rada et al., 2006, McBrien and Gentle, 2003, Elsheikh et al., 2010, Jobling et al., 2009, Kusakari et

al., 1997, Young et al., 2003, Yang et al., 2009)

Collagen

Types I, III, IV, V, VI, VIII, XII, & XIII (Jobling et al., 2009, Summers Rada et al., 2006, McBrien and Gentle, 2003, Kashiwagi et al., 2010, Wessel et

al., 1997, Yang et al., 2009, Kusakari et al., 1997, Trier, 2005)

Elastin (Moses et al., 1978, Maza et al., 2012)

Enzymes (McCluskey, 2001, McBrien and Gentle, 2003)

Fibroblasts & Myofibroblasts (Cui et al., 2008, McCluskey, 2001, Summers Rada et al., 2006, Young et al., 2003, McBrien and Gentle, 2003,

Cui et al., 2004)

Glycoproteins (McCluskey, 2001, Young et al., 2003, Tsai et al., 2010)

Growth Factors (Moring et al., 2007)

Proteoglycans (McCluskey, 2001, Tsai et al., 2010)

Aggrecan (McBrien and Gentle, 2003, Summers Rada et al., 2006, Rada et al., 1997)

Biglycan (McBrien and Gentle, 2003, Summers Rada et al., 2006, Rada et al., 1997)

Decorin (McBrien and Gentle, 2003, Summers Rada et al., 2006, Rada et al., 1997)

Glycosaminoglycans (Summers Rada et al., 2006)

Chondroitin sulfate (Summers Rada et al., 2006)

Dermatan sulfate (Summers Rada et al., 2006)

Heparan sulfate (Summers Rada et al., 2006)

Heparan (Summers Rada et al., 2006)

Hyaluronan (Kashiwagi et al., 2010, Summers Rada et al., 2006)

Keratan sulfate (Summers Rada et al., 2006)


26

1.6.3 Collagen

Mammalian scleral tissue is composed of approximately 90% collagen by weight (Summers

Rada et al., 2006, McBrien and Gentle, 2003, Yang et al., 2009). There are 8 collagen subtypes

present in human sclera: Type I, III, IV, V, VI, VIII, XII and XIII (McBrien and Gentle, 2003,

Summers Rada et al., 2006, Yang et al., 2009, McCluskey, 2001, Kashiwagi et al., 2010, Trier,

2005, Jobling et al., 2009, Kusakari et al., 1997). The structure and function of each collagen

subtype varies considerably (Summers Rada et al., 2006). Type I collagen fibres are the most

abundant type and can be present in up to 50‐70% of the human sclera (Yang et al., 2009).

With respect to diameters, adult human scleral collagen fibres are highly variable: fibrils

have an average range of 94‐102 nm and an overall range between 25‐250 nm (Summers

Rada et al., 2006). More specifically, the outer scleral layer – the episclera – has been

reported to have an average of 100 nm (Yang et al., 2009). In terms of thickness, the average

fibre ranges between 10‐25 μm (Tsai et al., 2010). During myopia development, the diameter

of the scleral collagen fibrils is reduced, weakening the sclera (Choo, 2003, Gentle et al.,

2003).

1.6.4 Elastin

As is characteristic of tissues that are subjected to multidirectional stretching, the human

sclera is partly composed of elastic fibres (McCluskey, 2001, Moses et al., 1978). Elastic fibres

are partly abundant in the lamina fusca and trabecular meshwork (Watson and Young,

2004); however, overall, elastic tissue forms less than 2% of the sclera per se (Moses et al.,

1978). The elastin is composed of non‐polar hydrophobic amino acids, such as alanine,

valine, isoleucine, and leucine, and contains little hydroxyproline and no hydroxylysine

(Watson and Young, 2004).

1.6.5 Enzymes

Notable enzymes present in the sclera are matrix metalloproteinases (MMPs), elastases,

collagenases, and proteoglycanases (Maza et al., 2012). MMPs, matrix‐degrading, zinc‐

dependent endopeptidases (Benoit de Coignac et al., 2000), are produced by scleral

fibroblasts (McCluskey, 2001) as either secreted or membrane‐bound pro‐enzymes or

zymogens (Cauwe et al., 2007). Remodelling and breakdown of the ECM occurs, at least in

part, by MMPs (McBrien and Gentle, 2003, Kashiwagi et al., 2010) since they are responsible

for degrading collagens, PGs, and other matrix macromolecules (Tomita et al., 2002): MMPs


27

cleave almost all ECM components (Pirilä et al., 2007). Other functions of MMPs are listed in

Table 1.6. The MMPs, of which there are 23 in humans (listed in Table 1.7), share a common

domain structure with a signal peptide for secretion, a propeptide, a catalytic domain, a

hinge region, and, in the majority of cases, a C‐terminal domain (Clark et al., 2008, Cauwe et

al., 2007). MMPs are activated by removal of the NH2‐terminal propeptide (Cauwe et al.,

2007). Altering the visual environment leads to changes in the concentration of MMPs

(Fredrick, 2002) and, accordingly, alterations have been observed in MMPs during myopia

development (McBrien et al., 2006). Specifically, levels of MMPs, and their tissue inhibitors,

increase during myopia development (Wride et al., 2006, Choo, 2003). MMP‐2 has been most

strongly implicated in the process of scleral remodelling (Jones et al., 1996).

Table 1.6: Known functions of MMPs. Some functions include regulation of cell growth,

regulation of apoptosis, and alteration of cell motility.

Functions of MMPs References

Opposing effects on angiogenesis via matrix degradation but

also release of angiogenesis inhibitors

(Clark et al., 2008, Cauwe et al.,

2007)

Regulation of cell growth via cleavage of cell surface‐bound

growth factors and receptors, release of growth factors

sequestered in the ECM or integrin signalling

Regulation of apoptosis via release of death or survival factors

Alteration of cell motility by revealing cryptic matrix signals,

or cleavage of adhesion molecules

Effects on the immune system and host defence

Modulation of the bioactivity of cytokines

Other proteases include elastases, collagenases, and proteoglycanases (McCluskey, 2001,

Maza et al., 2012). Elastase is regarded as a powerful proteinase that lacks specificity, unlike

some of the other proteinases, such as collagenase (Maza et al., 2012). Elastase, as its name

suggests, degrades elastin but, due to its lack of specificity, it is capable of degrading other

components of the ECM, such as collagen and PGs (Maza et al., 2012). Collagenase degrades

collagen fibrils; collagen degradation as a function in both normal and inflamed tissues

depends on the balance between collagenase and its inhibitors (Wooley, 1984).

Proteoglycanases degrade the core protein and link proteins of PGs (Maza et al., 2012).


28

Table 1.7: Known MMPs and their associated substrate specificity. In the context of myopia, MMP‐2, or gelatinase‐A, has been most strongly

implicated in the process of scleral remodelling.

Gene Name(s) Reference(s)

MMP‐1 Interstitial collagenase or fibroblast collagenase or collagenase‐1 (Tomita et al., 2002)

MMP‐2 Gelatinase‐A or 72kDa type IV collagenase (Benoit de Coignac et al., 2000, Clark et al., 2008)

MMP‐3 Stromelysin‐1 (Cauwe et al., 2007)

MMP‐7 Matrilysin‐1 (Benoit de Coignac et al., 2000, Clark et al., 2008)

MMP‐8 Neutrophil collagenase or collagenase‐2 (Pirilä et al., 2007, Giambernardi et al., 2001)

MMP‐9 Gelatinase‐B (Benoit de Coignac et al., 2000, Clark et al., 2008)



MMP‐12 Macrophage metalloelastase (Benoit de Coignac et al., 2000, Clark et al., 2008)

MMP‐13 Collagenase‐3 (Cauwe et al., 2007)

MMP‐14 Membrane‐type 1‐MMP (Clark et al., 2008)




MMP‐19 RASI‐1 (Cauwe et al., 2007)

MMP‐20 Enamalysin (Benoit de Coignac et al., 2000, Clark et al., 2008)

MMP‐21 Not classified (Cauwe et al., 2007)

MMP‐23 CA‐MMP (Cauwe et al., 2007)

MMP‐24 Membrane‐type 5‐MMP (Cauwe et al., 2007)

MMP‐25 Membrane‐type 6‐MMP (Cauwe et al., 2007)

MMP‐26 Matrilysin‐2 or endometase (Pirilä et al., 2007)

MMP‐27 ‐‐‐ (Clark et al., 2008)

MMP‐28 Epilysin (Cauwe et al., 2007)


29

1.6.6 Scleral Fibroblasts

Scleral fibroblasts – or sclerocytes – are present in the ECM, although not in abundance

(McCluskey, 2001), and are arranged in lamellae, or layers (Summers Rada et al., 2006,

Young et al., 2003). It is thought the lamella arrangement of the fibroblasts may play an

important role in controlling the size of the eye (Summers Rada et al., 2006). Activated

fibroblasts have a spindle‐shaped morphology with stress fibre formation, fibronectin fibrils

and focal adhesion complexes (Tomasek et al., 2002, Jester and Ho‐Chang, 2003, Jester et al.,

2002). Fibroblasts, along with the corneal stroma, are responsible for the production of

proteins found in the ECM, such as collagen, PGs, and hyaluronan (Kashiwagi et al., 2010,

McCluskey, 2001). Accordingly, the structural organisation and integrity of the sclera is

largely dependent on the activity of the scleral fibroblasts (McBrien and Gentle, 2003, Baglole

et al., 2006); fibroblasts both synthesise and remodel the ECM (Young et al., 2003). Scleral

fibroblasts are involved in scleral remodelling during axial elongation in myopia (Cui et al.,

2004, Cui et al., 2008) and it has been recently suggested that cellular signals acting on the

scleral fibroblast may direct the growth process resulting in myopia (Barathi et al., 2009).

Interestingly, the proliferation of fibroblasts in the sclera decrease during myopia

development but, during recovery, increase (Choo, 2003). Fibroblasts are thought to play a

role(s) during myopic development (refer to Figure 1.10).

Figure 1.10: Proposed model of the role of scleral myofibroblast cells in the biochemical and


30

biomechanical remodelling that facilitates myopia development (McBrien et al., 2009).

1.6.7 Glycoproteins

For the purposes of this document, the term ‘glycoprotein’ will be primarily used to describe

molecules consisting of an oligosaccharide with a mannose core N‐glycosidically linked to

asparagine; in the absence of the above clause, it could be argued that collagen, elastin, and

PGs are glycosylated proteins (Maza et al., 2012). Several non‐collagenous glycoproteins

have been characterised in the ground substance of the sclera during various embryological

developmental stages; they include fibronectin, vitronectin, and laminin (Summers Rada et

al., 2006). Fibronectin and laminin are also present in adult sclera (Chapman et al., 1998,

Fukuchi et al., 2001, Maza et al., 2012). It appears that fibronectin plays an important role in

the organisation of the pericellular and intercellular matrix due to its ability to bind to

collagen, fibroblasts, and glycosaminoglycans (GAGs) (Kleinman et al., 1981, Yamada et al.,

1980). Laminin, found in the basement membranes, participates in assembly of basement

membranes and promotion of cell adhesion, growth, migration, and differentiation

(Kleinman et al., 1985).

1.6.8 Growth Factors

It has been reported that many characteristics of scleral ECM remodelling are thought to be

under the control of specific growth factors (Summers Rada et al., 2006). During

embryological development, there are six known growth factors associated with scleral

growth: IGF‐I, IGF‐II, PDGF (platelet‐derived growth factor), TGF‐β (transforming growth

factor), NGF (nerve growth factor), and NE (norepinephrine) (Tripathi et al., 1991). It has

been observed in animal studies that, during myopia development, there is a decrease in the

levels of TGF‐β (Jobling et al., 2004). The levels of the growth factor, thrombospondin‐1

(Frost and Norton, 2007), have also been shown to reduce.

1.6.9 Proteoglycans

PGs comprise approximately 0.7‐0.9% of the total dry weight of the sclera (Summers Rada et

al., 2006). PGs, abundant ECM molecules, consist of a core protein with at least one attached

GAG side‐chain made up of repeating disaccharide units (Summers Rada et al., 2006,

McCluskey, 2001, Moreno et al., 2005, Ihanamäki et al., 2004). The GAG side‐chain attached

to the PG often carries a negative charge due to their high degree of sulfation (Summers


31

Rada et al., 2006, McCluskey, 2001). Scleral PGs serve several biologic functions: regulation

of hydration in vivo, maintenance of structural integrity, growth regulation, matrix

organisation, collagen fibril assembly and arrangement, and cell adhesion (Rada et al., 1997,

Summers Rada et al., 2006, McCluskey, 2001, Majava et al., 2007, Watson and Young, 2004).

PGs are synthesised from proteins produced and secreted by fibroblasts, as mentioned

previously (McCluskey, 2001, Johnson et al., 2006).

Glycosaminoglycans

As mentioned above, PGs consist of a core protein with at least one attached GAG side‐chain

made up of repeating disaccharide units. GAGs are constructed from several types of sugar

residues, which include N‐acetylglucosamine, N‐acetylgalactosamine, glucuronic acid, and

iduronic acid (Summers Rada et al., 2006). GAGs are categorised into four main groups

based upon their disaccharide composition, the type of linkage between disaccharides, and

the number and location of sulfate residues: 1) hyaluronan, 2) chondroitin sulfate and

dermatan sulfate, 3) heparan sulfate and heparan, and 4) keratan sulfate (Summers Rada et

al., 2006, Ihanamäki et al., 2004). The most abundant GAGs appear to be dermatan sulphate

and chondroitin sulphate; hyaluronic acid and heparan are only present in small amounts

(Maza et al., 2012).

Hyaluronan, a unique, non‐sulphated GAG, is an important constituent of the ECM: it plays

a role in regulating cell migration, proliferation, adhesion, development, and differentiation

(Kashiwagi et al., 2010, Laurent and Fraser, 1992, Knudson and Knudson, 1993). Hyaluronan

occupies the limited space between the collagen fibrils (Trier, 2005). Chondroitin sulphate

(consisting of sulphated N‐acetylgalactosamine and glucuronic acid), dermatan sulphate

(consisting of sulphated N‐acetylgalactosamine and two different types of uronic acid,

glucuronic acid and iduronic acid), heparan sulphate (sulphated N‐acetylglucosamine and

two different types of uronic acid, glucuronic acid and iduronic acid), and hyaluronic acid

(N‐acetylglucosamine and glucuronic acid) form the greater composition of the amorphous

ground substance present in the intercellular and interfibrillar spaces of the sclera (Maza et

al., 2012). Among the biochemical changes observed in myopic patients, decreased

production of GAGs has also been reported (McBrien et al., 2000, Rada et al., 2000). It must

also be noted that, as part of the aging process, there is a significant loss of GAG composition

(particularly dermatan sulphate); as GAGs are highly hydrated, the decrease in age‐related


32

tissue hydration can be attributed, at least in part, to this loss of GAG composition (Brown et

al., 1994).

Aggrecan, Biglycan, and Decorin

Three PG types have been identified in human sclera: aggrecan, biglycan, and decorin (Rada

et al., 1997, Johnson et al., 2006, Watson and Young, 2004, Boubriak et al., 2003). Of the PGs

synthesised from sclera organ culture, aggrecan, biglycan, and decorin represent 6%, 20%,

and 74% respectively (Trier, 2005, Rada et al., 1997). Compared to biglycan and decorin,

aggrecan is a comparatively large PG: it contains over 100 chondroitin sulfate chains, over 30

keratan sulfate chains (Rada et al., 1997), and more than 50 oligosaccharide chains (Vynios et

al., 2001). Aggrecan has a space filling role in both cartilage and the sclera (Fullwood et al.,

2011). Biglycan – also referred to as PG I – is a small PG (McBrien and Gentle, 2003); it has a

molecular mass of 200 kDa (Rada et al., 1997). Biglycan contains two chondroitin‐dermatan

sulfate chains (Rada et al., 1997, Vynios et al., 2001) and belongs to the Small Leucine‐Rich

Proteoglycans (SLRPs) family (Summers Rada et al., 2006, Zhang et al., 2009). SLRPs are

characterised by a central domain containing leucine‐rich repeats flanked on both N‐ and C‐

terminal sides by small cysteine clusters (Moreno et al., 2005, McEwan et al., 2006). Biglycan

plays an important role in regulating collagen fibril assembly and interaction (McBrien and

Gentle, 2003, Zhang et al., 2009). Decorin, the most abundant PG (Trier, 2005), is reasonably

small: it consists of a molecular mass of 120 kDa (Rada et al., 1997). Decorin contains one

chondroitin‐dermatan sulfate chain (Rada et al., 1997, Vynios et al., 2001). Biglycan and

decorin are closely related (Rada et al., 1997, McCluskey, 2001); decorin also belongs to the

SLRP family (Summers Rada et al., 2006, Zhang et al., 2009). [Both biglycan and decorin are

known as Class I SLRPs (Schaefer and Iozzo, 2008).] Decorin exhibits a unique organisation

of 10 tandem leucine‐rich repeats enabling the molecule to fold into an arch‐shape structure

suited to bind both globular and non‐globular proteins (Santra et al., 2002). Decorin – also

referred to as PG II – binds, in vitro, to collagens type I and II (Rada et al., 1997) and, along

with biglycan, plays an important role in collagen fibril assembly and interaction (McBrien

and Gentle, 2003, Zhang et al., 2009).

1.6.10 Other Receptor Types known to be Present in Human Sclera

Several receptor types have been found in human sclera; refer to Table 1.8. There has been

interest in muscarinic receptors as a potential target for pharmaceutical agents, in the context


33

of myopia, but the studies have found variable effects (Luft et al., 2003, Diether et al., 2007).

Table 1.9 lists known locations of muscarinic receptors in both ocular and non‐ocular tissues

and Table 1.10 outlines key functions of receptors shown in Table 1.8.

Table 1.8: Receptor types observed in human sclera. Types include collagen, prostanoid,

and muscarinic receptors.

Receptor Type Present in Human Sclera References

α1(XIII) Collagen mRNA (Sandberg‐Lall et al., 2000)

ADORA1 (Cui et al., 2008)

ADORA2B (Cui et al., 2008)

CD14 (Chang et al., 2004)

E‐Prostanoid Receptors (Subtypes 1‐4) (Schlötzer‐Schrehardt et al., 2002)

FP (PGF2α Receptor) (Schlötzer‐Schrehardt et al., 2002)

MD‐2 (Chang et al., 2004)

Muscarinic Receptor Subtype 1 (M1) (Collison et al., 2000, Qu et al., 2006)

Muscarinic Receptor Subtype 2 (M2) (Qu et al., 2006)

Muscarinic Receptor Subtype 3 (M3) (Collison et al., 2000, Qu et al., 2006)



TLR4 (Chang et al., 2004)

Table 1.9: Location of muscarinic receptors in ocular and non‐ocular tissues.

Location Muscarinic Receptor/s Reference

Cornea and epithelium of

the crystalline lens

M3 (Collison et al., 2000)

Iris sphincter and ciliary

body

M1‐M5 (Gil et al., 1997, Gupta et al.,

1994, Zhang et al., 1995)

Retina M3 and M4 (Fischer et al., 1998)

Lung Fibroblasts M1‐M4 (Matthiesen et al., 2006)

Labial Salivary Glands M1, M3, M5 (Ryberg et al., 2008)

Sclera M1‐M5 (Qu et al., 2006)


34

Table 1.10: Key functions of receptors present in human sclera.

Role or Possible Involvement Reference

ADORA1 and ADORA2B Receptors

Associated with the regulation of IOP in rabbits and monkeys (Crosson et al., 1994, Tian et al.,

1997)

ADORA1 inhibits adenylcyclase (Gi/o‐coupled) and therefore

decreases levels of cAMP (cyclic adenosine monophosphate)

(Burnstock, 2007)

ADORA2B activates adenylcyclase (Gs‐coupled) and increases

levels of cAMP

(Burnstock, 2007)

CD14 Receptors

Enhances activation of cells by lipopolysaccharide (Viriyakosol et al., 2000)

E‐Prostanoid Receptors

Exert a variety of actions in various tissues and cells; some of these

actions are described below:

relaxation and contraction of various types of smooth

muscles

modulate neuronal activity by either inhibiting or stimulating

neurotransmitter release, sensitizing sensory fibers to

noxious stimuli, or inducing central actions such as fever

generation and sleep induction

involved in apoptosis, cell differentiation, and oncogenesis

regulate the activity of blood platelets both positively and

negatively and are involved in vascular homeostasis and

haemostasis

(Narumiya et al., 1999)

FP (PGF2) Receptors

May play a role in regulating IOP (Schlötzer‐Schrehardt et al.,

2002)

MD‐2 Receptors

Accessory molecule that associates with the extracellular domain of

TLR4 (see below for more on TLR4) conferring on it

lipopolysaccharide responsiveness

(Nagai et al., 2002, Shimazu et

al., 1999)

Muscarinic Receptors

Transactivate growth factor receptors (Kanno et al., 2003)

Accommodation, altered aqueous outflow, and contraction of the

iris sphincter muscle

(Gabelt and Kaufman, 1992,

Kaufman, 1984)

Regulating growth of the corneal epithelium and control corneal

wound healing

(Lind and Cavanagh, 1995)

TLR4 Receptors

Recognition of the highly conserved pathogen‐associated

molecular patterns unique to microbes

(Chang et al., 2004)

Stimulation results in the activation of an immunostimulatory and

immunomodulatory cell‐signalling pathways essential for innate

immunity and activation of the adaptive arm of the immune

response

(Medzhitov et al., 1997, Schnare

et al., 2001)

1.6.11 Factors Affecting Human Scleral Structure & Function

During myopia development, fibril diameter and the processes involved in collagen

synthesis and degradation change (McBrien et al., 2006). In a human myopic eye, the sclera

thins and the diameter of collagen fibrils found in the posterior sclera becomes progressively,


35

as compared to non‐myopic eyes, smaller (Jobling et al., 2009, Kusakari et al., 1997, Cui et al.,

2008, McBrien et al., 2001a, McBrien et al., 2000, Tseng et al., 2004, Yang et al., 2009, Gottlieb

et al., 1990). The thinning of the sclera and the narrowing of the fibrils is usually associated

with a disconnection of the collagen fibre bundles (Yang et al., 2009) and an abnormal

pattern of interlacing (Gottlieb et al., 1990). It has been hypothesised that the above‐

mentioned pathological features of the fibrils – narrowing and disconnection – results from

reduced collagen synthesis and/or increased collagen degradation (Yang et al., 2009).

Accordingly, elevated activity of collagen‐degrading enzymes has been implicated in early

stages of myopia onset (McBrien et al., 2001a). The process of scleral remodelling is depicted

in Figure 1.11.

Figure 1.11: Diagrammatic representation of the scleral remodelling that underpins myopia

development (McBrien et al., 2009). It is believed that the visually‐generated signal is

communicated from the retina and through the choroid to the sclera.

1.7 Summary

Based on the literature described here, it is clear that distribution and function of GABA in

scleral tissue is a relatively unexplored area. Previously, the human sclera has been thought

to be essentially inert (McBrien and Gentle, 2003) and, accordingly, there is a significant gap

in the literature addressing and exploring the potentially dynamic role(s) of the sclera –

including in the context of communication with neurotransmitters, such as GABA – in

refractive conditions (Watson and Young, 2004). GABA is an important inhibitory


36

neurotransmitter present in the CNS and retina (Krogsgaard‐Larsen et al., 1997, Chebib et al.,

2009b, Enz and Cutting, 1999, Barnard et al., 1998). Specifically, GABA antagonists have

been found to influence ocular growth in chick models of myopia (Stone et al., 2003, Leung et

al., 2005) and, in association with inhibition of myopia development in chicks, facilitate

learning and memory in mice (Chebib et al., 2009b). One key animal study (Stone et al.,

2003) demonstrated that GABA plays a modulation role during eye growth and refractive

development. As stated in a recent paper (Cheng et al., 2011), due to the fact that there are

many examples of GABA receptors located in multiple ocular sites, the discovery of rho 1

GABAC receptors in non‐neuronal ocular tissues is not as unusual as it may first appear.

This study will provide important new information on GABA receptor localisation in human

scleral tissue at both a whole of tissue and cellular level.

We hypothesise, based on the limited chick data available, that GABA receptors will be

present in human non‐retinal tissues, including RPE and sclera.


37

1.8 Research Aims

The aims of this research project were:

1) Investigate the expression of GABA receptors in human scleral tissue using both

immunohistochemistry (IHC) and gene expression studies

2) Investigate the expression of GABA receptors in cultured human scleral cells by

culturing scleral cells ex vivo and examining these for GABA receptor gene expression

3) Investigate the expression of GABA receptors in choroidal, RPE, and retinal tissue

using both IHC and gene expression studies

The overarching theme of this thesis was to investigate human sclera – cells and tissue – and

factors that may impact scleral cell growth with relevance to myopia. The first study to be

completed was to determine if GABA receptors are present in human scleral tissue. Based

on the fact that these receptors have been localised to the scleral tissue of other species

(Cheng et al., 2011), it was predicted that GABA receptors would also be present in human

scleral tissue. The second study sought to target mesenchymal stromal cell populations

obtained from human scleral cell cultures. Thirdly, we investigated the potential presence of

GABA receptors in choroidal, RPE, and retinal tissues obtained from the same donors as the

scleral tissue and stromal cells were sourced.


38

2 Materials and methodology

This project utilised IHC – antibodies raised against four different GABA receptor subunits

previously found in human retina – and reverse‐transcriptase polymerase chain reaction

(RT‐PCR) – 21 different primers designed to amplify all known GABA receptor subunits.

IHC was only undertaken using human scleral tissue; RT‐PCR was undertaken using human

sclera, choroid/RPE, retina, and cultured human scleral cells. The following sections outline

all materials used and methodology implemented during this project and experiments are

listed in the following order: Experiment (i) – IHC; Experiment (ii) – Cell culturing and flow

cytometry (Dominici et al.) ; & Experiment (iii) – Analysis of GABA receptor gene expression

in ocular tissues and cells.

2.1 Human Research Ethics Issues

This project involves the use of human cadaveric tissue and, therefore, ethics approval was

required. For this project, the Queensland University of Technology (QUT) ethics committee

granted ethics approval for all experimental work. The QUT ethics approval number for this

project is 0800000807.

2.2 Eye Collection

Eye cups were collected from the Queensland Eye Bank (QEB), Princess Alexandra Hospital,

Brisbane, Queensland within 4‐5 days of death. Until collection, the tissue was stored at 4C

in tissue storage medium. Since eye cups were obtained in pairs, the right eye was fixed in

formalin for 2 days and then stored in 70% ethanol at 4°C; the right eye was used for IHC.

The left eye was stored at ‐80°C for subsequent gene expression studies. The age of the

donors varied from 61 to 86 years. The refractive history and ethnicity of the donors was not

known. However, the age and cause of death of all donors was known (Table 2.1).

Table 2.1: Donor characteristics; information supplied from the QEB.

Age Cause of Death IHC or RT‐PCR

71 Unknown IHC

86 Non‐Small Cell Lung Cancer IHC

79 Intracranial Haemorrhage IHC

65 Metastatic Lung Cancer RT‐PCR

61 Metastatic Pancreatic Cancer RT‐PCR

75 Lung Adenocarcinoma, COPD, Hypertension RT‐PCR


39

2.3 Preparation for Microtomy

Whole eyes were sectioned into strips from the inferior, superior, medial, and lateral

quadrants ~15 mm x 5 mm from the anterior section to approximately 3 mm from the start of

the optic nerve (Figure 2.1 shows a photograph of a typical eye specimen). The rationale for

obtaining the four different regions was to allow for potential variability (at a cellular level)

from one region to another. Orientation (Figure 2.2 maps out directional orientation and

Figure 3 provides an illustration depicting how the eyes were dissected) was determined

using several parameters: as it was already known whether the eye was left or right

(supplied information from QEB) and the eyes each presented with a reasonable length of

optic nerve, it was relatively straightforward to determine the general orientation of the eye

based on the direction of the optic nerve. More specifically, the vortex veins were visible and

served as landmarks for determining the orientation. In some cases, the rectus and oblique

muscles were attached as well. For more information on the gross anatomy of human sclera

and associated landmarks, refer to Appendix 2.

Figure 2.1: Photograph (taken by EFM) of a typical eye specimen showing length of the optic

nerve and visible veins. In the top left region of the eye (as shown in the picture), some

remnants of muscle are evident.


40

Figure 2.2: Scleral map highlighting superior, inferior, nasal, and temporal regions in

relation to the optic nerve (Elsheikh et al., 2010).

Figure 2.3: Illustration demonstrating how the eye cups were dissected. Each eye was cut

into four strips – superior, inferior, medial and lateral. Each strip was approximately 5 mm

wide and 15 mm in length.

2.4 Microtomy

The formalin‐fixed tissue sections mentioned above were processed by routine paraffin

embedding technique. The processing (dehydration with alcohols, clearing with xylene, and

infiltration with molten wax) was performed using a Tissue‐Tek VIP 1000 by Helen

OʹConnor at QUT, Gardens Point. Embedding was undertaken using a Tissue‐Tek Tissue

Embedding Console System 4593. Microtomy was performed using either a Reichert‐Jung

2030 (Queensland Eye Institute, or QEI) or a Microm HM 325 (QUT, Gardens Point) – both

are rotary microtomes with disposable blades. Serial sections were cut to 3 microns in width

and mounted on glass slides (Menzel HD Microscope Slides, Superfrost Plus Ground).

Tissue sections were dried at 60°C for several hours.

2.5 Preliminary Work: H&E and Bleaching Optimisation

The paraffin tissue sections were deparaffinised using xylene and rehydrated using

ascending grades of ethanol (70, 90, and 100%). Haematoxylin & eosin staining was

undertaken to examine the general morphology of the tissue (refer to Figure 2.4 for H&E


41

images – donor 6397L – showing the landscape of the sclera). The tissue was stained with

Mayerʹs haematoxylin for 3 minutes, differentiated with two dips in acid alcohol, and treated

with Scottʹs reagent for 30 seconds. The images were taken using an Olympus BX41

microscope and a Nikon DXM 1200 camera.

Figure 2.4: Overview of scleral tissue structure, including demonstration of melanocytes in


42

deeper sclera, from donor 6397L to determine integrity of tissue and basic structures using

H&E.

(A) A montage of three photographs (20x oil immersion lens) demonstrating nearly the full

thickness of the sclera and adjacent retinal tissue in a lateral section. Note the higher density

of stromal cell nuclei within the outer third of the sclera along with the presence of blood

vessels. (B) A corresponding image of the outer sclera within a section taken from the

medial aspect of the sclera. Note the thicker muscular wall of the arteriole surrounded by

unexpected melanocytes. (C) A higher power image (100x oil immersion lens) showing the

detailed morphology of the vessel wall (the arrow is highlighting the endothelium) and

adjacent melanocytes, which appear brown (medial section).

As can be seen in Figure 2.4 above, there are melanocytes present in the sclera. Due to the

possibility of melanocytes being mistaken for false‐positive antibody results (Orchard and

Calonje, 1998), bleaching – exposure to oxalic acid for one minute and to potassium

permanganate for 3‐5 minutes – was attempted prior to undertaking IHC. It was found that

3‐5 minutes of bleaching was an optimal timeframe to ensure the melanocytes appeared pale

instead of brown (refer to Figure 2.5). However, bleaching the tissue also increased the

basophilia of the tissue and resulted in a bluish appearance.

Figure 2.5: Bleaching optimisation results observed for donor 6397L (tissue taken from

lateral region).

(A) Mayer’s haematoxylin only as a control; the arrow is highlighting a melanocyte (B)

Appearance of tissue following one minute of oxalic acid and 30 seconds of potassium

permanganate. As shown, bleaching increased the basophilia of the tissue and resulted in

the tissue becoming negatively charged. The pigmentation has started to disappear but not

entirely; the arrow is highlighting the outer layer of sclera; (C) Appearance of tissue

following one minute of oxalic acid and 1 minute of potassium permanganate. Bruch’s

membrane has started to become visible and there is still pigmentation present; (D)

Appearance of tissue following one minute of oxalic acid and 2 minutes of potassium

permanganate. Still a small amount of pigmentation visible; (E) Appearance of tissue

following one minute of oxalic acid and 5 minutes of potassium permanganate. Bruch’s

membrane is now well‐defined as a thin blue line beneath the RPE.


43

2.6 Immunohistochemistry: Experiment #1

IHC was performed using the Dako EnVision+ HRP (DAB) kit (DakoCytomation, Botany,

New South Wales, Australia). The following antibodies were used:

1) QED Biosciences Inc. Anti‐Alpha 1 GABA‐A Receptor Monoclonal (Lot # 032510)

2) Sigma Aldrich Prestige Antibodies Anti‐GABRR2 Rabbit (Lot # R06608)

3) Sapphire Biosciences Rb pAb to GABRR1 (Lot # GR25385‐2)

4) Epitomics GABA (B) R2 Rabbit mAb (Lot #YE102203)

The antibodies listed above – excluding the one for GABA B R2 – were selected based on

previous studies demonstrating presence in human retinal tissue: GABA A α‐1 (Vardi and

Sterling, 1994), GABA 1 (Gussin et al., 2011, Hackam et al., 1997), and GABA 2 (Wang et

al., 1994, Hackam et al., 1997). GABA B R2 was selected as representative for the GABA B

receptor type. Deparaffinised slides were blocked in 2% bovine serum albumin (BSA) in

phosphate buffered saline (PBS) for 30 minutes. The primary antibodies were diluted in 2%

BSA (1:100 and 1:200) and applied at room temperature for one hour. The peroxidise‐

labelled polymer was added for 20 minutes at room temperature after the slides were

washed three times in PBS to remove the primary antibody. DAB, or 3,3‐diaminobenzidine,

was added for 5 minutes and then the slides were counterstained with Mayer’s

haematoxylin. The sections were dehydrated using descending grades of ethanol (100, 90, &

70%), followed by xylene, and then mounted using Entellan New® (Pro Sci Tech,

Thuringowa, Queensland, Australia). Sections were viewed with an Olympus BX 41

microscope and images were taken using a NikonDXM 1200 camera. [All four quadrants

were exposed to all four antibodies using donor 6937L; the superior and medial sections

were exposed to the Anti‐Alpha 1 GABA‐A Receptor Monoclonal (QED Biosciences Inc) and

the Anti‐GABRR2 (Sigma Aldrich Prestige Antibodies) using donors 6806R and 6813R.]

2.7 Cell Culturing & Flow Cytometry: Experiment #2

Scleral stromal cells obtained from donors 6952 L and 6953L were kindly provided by Assoc

Prof Damien Harkin from QEI and characterised using FC by Dr Laura Bray (also from QEI).

An explant of scleral tissue – 1 cm2 – was washed twice in PBS, chopped finely into 1 mm2

pieces, resuspended in 2 ml fetal bovine serum (FBS), and seeded into a 25 cm2 flask and the

pieces were distributed. The flask was inverted and incubated for 5 hours at 37°C in a cell

culture incubator (5% CO2) to promote attachment of tissue pieces. The flask was then

reverted to normal orientation and 2 mL of culture medium (DMEM/F12 with 10% FBS and


44

10 mM glutamine, supplemented with 50 U/ml penicillin/50 μg/ml streptomycin antibiotic

solution) was added. Out‐growth of stromal cells was clearly visible using a light

microscope after 1 week. The cultures were expanded to passage 3 (over a period of 5‐6

weeks) before storage in liquid nitrogen; 7.5 million cells per donor (stored in 90% FBS + 10%

DMSO).

Cellular phenotype analysis was performed using fluorescently labelled mouse anti‐human

antibodies (or rat anti‐human antibody in the case of CD44) as follows: IgG1 к isotype

control phycoerythrin (PE) (MOPC‐21), CD31 PE (WM‐59, IgG1 к), CD34 fluorescein

isothiocyanate (FITC) (581/CD34, IgG1 к), CD44 Pacific Blue (IM7, IgG2b, к), CD45 PE‐

cyanine dye (Cy7) (HI30, IgG1 к), CD73 allophycocyanin (APC) (AD2, IgG1 к), CD90 FITC

(5E10, IgG1 к), CD105 PE (SN6, IgG1 к), and CD141 PE (1A4, IgG1 к). The panel of markers

was a standard panel used to determine mesenchymal‐like characteristics. All antibodies

were purchased from Becton‐Dickinson (BD Pharmingen, CA, USA), eBioscience (in the case

of CD73 and CD105; Jomar Bioscience, SA, Australia), or Biolegend, Australian Biosearch,

WA, Australia (in the case of CD44). Viability was assessed by 7‐amino‐actinomycin D

incorporation (BD Viaprobe cell viability solution). Antibody concentrations were used

according to manufacturers’ instructions. Purified antibodies against GABAA‐α1, GABAAB‐

2, GABR‐R1, and GABR‐R2 (same as was used for IHC) were also utilised with either a

secondary Alexa Fluor 488 (for anti‐mouse) or 594 (for anti‐rabbit) antibody. Forty thousand

events were collected on a BD LSRII (BD Biosciences, CA, USA) where possible and analysed

using FlowJo software (Tree Star Inc., OR, USA).

2.8 Analysis of GABA Receptor Gene Expression in Ocular Tissues and Cells: Experiment #3

2.8.1 Primer Design

Primers were designed to amplify ~150‐200 base pair (bp) fragments of individual GABA

receptors sequences. Individual primers were 21 nucleotides in length and, of the total

number, 12‐13 nucleotides consisted of guanine or cytosine. Database searches using

determined sequences were performed using the BLAST program to confirm specificity of

the primers to their target GABA receptors. The sequences were aligned by using

ClustalW2. The following table (Table 2.2) lists the primers. Primers were obtained from

Sigma Aldrich. As the annealing temperature had to be deduced for each of the primers, the


45

primers were exposed to three different temperatures: 63.0°C, 65.0°C, and 67.0°C. Analysis

for each primer was performed at the three temperatures mentioned and, if expression was

observed at a unique temperature, that temperature was determined to be the annealing

temperature. The results from the temperature‐range optimisation are shown in Table 2.3.

The remaining four primers not shown in the table – GABAA Alpha 2 v1 & 2, GABAA Alpha

3, GABAA Delta, and GABAC Rho 3 – did not demonstrate expression.


46

Table 2.2: Individual primers (and expected product size) for each gene, including all known

transcript variants where applicable and represented by a ‘V’ in the gene name, representing

a GABA receptor subunit. The primer for GAPDH (housekeeping gene) is also shown below.

# GABA Gene Primer Product (bp)

1 GABAAΑ1V1,2,3,4,5,6,7

(GABA A ALPHA 1 V1‐V7)

F460 5ʹ AGCCCGCGATGAGGAAAAGTC – 3’

R653 5ʹ CCCAATCCTGGTCTCAGGCGA – 3’

193

2 GABAAΑ2V1 & 2

(GABA A ALPHA 2 V1 & V2)

F1340 5’ GGGCTTGGGATGGGAAGAGTG – 3’

R1504 5’ TGGGTTCTGGCGTGGTTGCAC – 3’

164

3 GABAAΑ3

(GABA A ALPHA 3)

F512 5’ TGGGCTTGGAGATGCAGTGAC – 3’

R703 5’ TGTGGAAGAAGGTGTCCGGTG – 3’

191

4 GABAAΑ4V1‐3

(GABA A ALPHA 4 V1‐V3)

F1867 5’ CTGTCCTCACCATGACCACAC – 3’

R2072 5’ CTCTCTCTGCACTGGAGCAGC – 3’

205

5 GABAAΑ5V1 & 2

(GABA A ALPHA 5 V1 & V2)

F746 5’ ATCACTCAGGTGAGGACCGAC – 3’

R929 5’ TGTCTGGGGTCCAGATCTTGC – 3’

183

6 GABAAΑ6

(GABA A ALPHA 6)

F609 5’ AGGTTGAAGTTTGGGGGGCCA – 3’

R801 5’ AACCAGCCTCATGGGACAGTC – 3’

192

7 GABRB1

(GABA A BETA 1)

F561 5’ GTTGGGATGCGGATCGATGTC – 3’

R731 5’ TGGTACCCAGAGTTGGTCAGC – 3’

170

8 GABRB2V1 & 2

(GABA A BETA 2 V1 & V2)

F1765 5’ CCTCCCACTGGAAGCAAGGAC – 3’

R1897 5’ TGGACCACAGGATAGGCCAGC – 3’

132

9 GABRB3V1‐4

(GABA A BETA 3 V1‐V4)

F2647 5’ GAGGCAAAGGAGGGCAACCTG – 3’

R2774 5’ GCGTCTATGAAACCGGTGCAC – 3’

127

10 GABRG1

(GABA A GAMMA 1)

F816 5’ AAGCCCTCCGTAGAAGTGGCT – 3’

R1064 5’ CGATGTTCTTGCAGGCACTGC – 3’

248

11 GABRG2V1,2,3

(GABA A GAMMA 2 V1‐V3)

F74 5’ TCTCCTTGTACCCTCCCCCTG – 3’

R257 5’ CCACCTGCCTCTAGTAGGTCC – 3’

183

12 GABRG3

(GABA A GAMMA 3)

F1094 5’ TGCTACGCCAGCAAGAACAGC – 3’

R1242 5’ CGGCGAAGACAAACAGGAAGC – 3’

148

13 GABRE

(GABA A EPSILON)

F361 5’ GTCAACAGCCTTGGTCCTCTC – 3’

R579 5’ GACCATCTGGTTGGGCATGGT – 3’

218

14 GABRD

(GABA A DELTA)

F385 5’ ACAGCAGGCTCTCCTACAACC – 3’

R577 5’ GTGGAGGTGATTCGGATGCTG – 3’

192

15 GABRQ

(GABA A THETA)

F404 5’ GACCCTGGACTATCGGATGCA – 3’

R559 5’ GATCCAGGGAACAAGCTGCTG – 3’

155

16 GABRP

(GABA A PI)

F452 5’ CCATATACCTCCGACAGCGCT – 3’

R657 5’ TCTGAGGGCATACAGGACCGT – 3’

205

17 GABBR1, 2, & 3

(GABA B R1)

F928 5’ GACGTGAATAGCCGCAGGGAC – 3’

R1122 5’ GAGGTTCCACATCCTAGCAGC – 3’

194

18 GABBR2

(GABA B R2)

F2900 5’ ATGCAGCTGCAGGACACACCA – 3’

R3081 5’ GTTCGAGAGGGCTCTGTTGTG – 3’

181

19 GABRR1

(GABA RHO 1)

F826 5’ TGAGGCACTACTGGAAGGACG – 3’

R981 5’ CGTTGTCTGTGGTGGTGTCGT – 3’

155

20 GABRR2

(GABA RHO 2)

F380 5’ TATGACCCTGTACCTGCGGCA – 3’

R581 5’ CACGTGTCCATCTGGGAACAC – 3’

201

21 GABRR3

(GABA RHO 3)

F339 5’ GAGACCTGGATTTGGAGGGTC – 3’

R491 5’ GCTGTGCTAGGAAAGGAGAGC – 3’

152

22 GAPDH F 5’ GCAAATTCCATGGCACCGT – 3’

R 5’ TCGCCCCACTTGATTTTGG – 3’

106


47

Table 2.3: Annealing temperatures for primers following exposure of all cells and tissues to

three different temperature ranges: 63°C, 65°C & 67°C.

Annealing Temperature Primer Temperature/Primera

63°C GABA A Alpha 1 v1‐7 F: 70.5°C

R: 72.7°C

GABA A Alpha 5 v1 & 2 F: 65.5°C

R: 68.6°C

GABA A Beta 1 F: 70.8°C

R: 66.3°C

GABA A Beta 2 v1 & 2 F: 69.2°C

R: 70.5°C

GABA A Gamma 2 v1‐3 F: 68.4°C

R: 63.8°C

GABA A Gamma 3 F: 69.7°C

R: 70.0°C

GABA A Epsilon F: 65.1°C

R: 70.3°C

GABA A Theta F: 68.3°C

R: 68.1°C

GABA A Pi F: 67.1°C

R: 67.9°C

GABA B R2 F: 71.6°C

R: 66.1°C

GABA Rho 1 F: 66.5°C

R: 68.5°C

GABA Rho 2 F: 69.6°C

R: 67.8°C

65°C GABA A Alpha 4 v1‐3 F: 65.6°C

R: 66.9°C

GABA A Alpha 6 F: 72.4°C

R: 67.5°C

GABA A Beta 3 v1‐4 F: 71.0°C

R: 68.7°C

67°C GABA A Gamma 1 F: 67.1°C

R: 70.2°C

GABA B R1 F: 69.9°C

R: 64.9°C a Information supplied by the manufacturer.

2.8.2 RNA Isolation and Reverse‐Transcription

RNA was isolated from tissue samples and cultured scleral cells using TRIzol solution

(Invitrogen). In the case of the tissue samples, each layer was separated. Firstly, the retina

was carefully loosened from the choroid and RPE. Due to difficulty separating the choroid

from the RPE, both layers remained intact. Finally, the sclera was cleaned of any

contamination – easily observed with the eye due to the difference in colours of the layers.

Briefly, tissues were ground to a fine powder under liquid nitrogen using a mortar and

pestle and this was then transferred to a 2 ml Eppendorf tube containing 1 ml TRIzol. Total


48

RNA was extracted from this following the manufacturer’s recommendations. The cells

were allowed to incubate at room temperature for five minutes. Cell homogenate was

transferred to 1.5 mL Eppendorf tubes. Two hundred L of chloroform was added to each 1

mL cell homogenate. Tubes were mixed vigorously for 15 seconds and then allowed to

stand for 2 minutes. Tubes were spun in the cold room (4C) for 15 minutes at 12,000 rpm.

The upper aqueous phase was carefully transferred to new Eppendorf tubes. RNA was

precipitated by adding 0.5 mL isopropyl alcohol per 1 mL of tissue homogenate. Tubes were

incubated for 10 minutes at room temperature. Tubes were spun at 12,000 rpm at 4C for 10

minutes. Supernatant was carefully discarded and the pellet was washed with 1 mL of 75%

ethanol per 1 mL of supernatant and mixed well. Tubes were spun at 7,500 rpm for 5

minutes. The supernatant was carefully removed and discarded. The pellet was

resuspended in 30 L of de‐ionised water and the pellet was mashed finely using the tip of a

sterile pipette. The RNA was quantitated using a NanoDrop Spectrophotometer (Thermo

Scientific, Wilmington, DE) and 2 g of total RNA was reverse‐transcribed for 10 minutes at

25°C, 60 minutes at 55°C, and 15 minutes at 70°C using 3 μl pD(N)6 primers (Invitrogen), 200

μM each deoxyribonucleotide triphosphate (dNTP) (Pharmacia, Uppsala, Sweden) and 200

U Superscript III reverse transcriptase (Invitrogen); the total reaction volume was 10 μL.

The concentration and purity of the RNA was determined by measurement of the optical

density at 260 nm and 280 nm and the results are shown in Table 2.4. The 260/280 nm ratios

ranged between 1.17 ‐ 1.99 but were on average 1.67. Lower ratios were seen in samples

from frozen tissues and, in particular, those extracted from the retina, which produces retinal

pigments, including melanin. Furthermore, it is important to note that the 260 nm/230 nm in

the retinal samples were influenced by the melanin found in these tissues but the RNA was

still suitable for expression analysis via RT‐PCR. The integrity of the cDNA made using the

RNA extracted from these samples was confirmed using primers to amplify the

housekeeping control gene, GAPDH. Attempts were made to extract RNA from donors

6806L, 6813L, and 6815L but the RNA yields were too small to complete the analysis of gene

expression for all of the GABA receptors in these samples – between 35.3 and 653.2 ng/μL –

so were excluded from the project.


49

Table 2.4: Results from NanoDrop.

Sample Identification Concentration RNA ng/μl 260/280nm 260/230nm

6952L A Sclera 1862.8 1.46 0.77

6952L B Sclera 2902.3 1.8 1.16

6952L C Sclera 3311.7 1.67 1.31

6952L D Sclera 2502.0 1.55 1.02

6952L E Sclera 2295.2 1.56 0.92

6952L F Sclera 2763.5 1.70 1.10

6953L A Sclera 2201.8 1.64 0.89

6953L B Sclera 2677.8 1.37 1.08

6953L C Sclera 2467.0 1.66 0.99

6953L D Sclera 2551.4 1.65 1.02

6954L A Sclera 2455.5 1.77 0.99

6954L B Sclera 2981.6 1.60 1.19

6954L C Sclera 1346.3 1.25 0.57

6954L D Sclera 2303.9 1.69 0.92

6952L A Mesenchymal Stromal Cells 868.3 1.95 1.50

6952L B Mesenchymal Stromal Cells 911.8 1.98 1.88

6952L C Mesenchymal Stromal Cells 892.2 1.98 1.82

6952L D Mesenchymal Stromal Cells 1042.13 1.96 1.84

6953L A Mesenchymal Stromal Cells 1058.5 1.96 1.69

6953L B Mesenchymal Stromal Cells 1244.7 1.99 1.86

6953L C Mesenchymal Stromal Cells 952.6 1.95 1.63

6953L D Mesenchymal Stromal Cells 1087.6 1.97 1.95

6952L A Retina 3688.0 1.62 1.45

6952L B Retina (excluded) 281.2 1.43 0.72

6953L A Retina 3583.5 1.18 1.42

6952L A Choroid/RPE 2915.8 1.64 1.19

6953L A Choroid/RPE 3438.4 1.48 1.40

6953L B Choroid/RPE 3182.1 1.17 1.86

2.8.3 RT‐PCR Amplification of GABA Receptors

Primers specific to all 21 GABA receptor subunits (as shown previously in Table 2.2) and the

housekeeping gene GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase) were used to

analyse gene expression. RT‐PCR reactions were performed in a PTC‐200 thermocycler

(Perkin Elmer, Waltham, MA) under the following conditions: Two μl cDNA template, 1.5

mM MgCl2, 200 μM each dNTP, 50 ng of each forward and reverse primer, and 0.5 units of

Platinum Taq polymerase in 1 x PCR buffer (Invitrogen); the total reaction volume was 50

μL. Cycling conditions included an initial denaturation at 94°C for 2 minutes, followed by


50

40 cycles at 94°C for 30 seconds, 63‐67°C for 30 seconds and 72°C for 30 seconds, with a final

extension of 72°C for 7 minutes. Amplification products were visualised by ethidium

bromide staining following separation by electrophoresis through 2% agarose gels. The

housekeeping gene GAPDH was used to confirm the integrity of the cDNA. Controls used:

“PCR‐ve” – water was substituted for template; “housekeeping control” – primer used was

for housekeeping gene to confirm integrity of the cDNA sample; “RT‐ve” – Superscript III

was not added during reverse‐transcription. The number times a RT‐PCR reaction was

performed was once (following optimisation of the annealing temperatures). Purified RT‐

PCR product for each representative GABA gene was sent to the Australian Genome

Research Facility (AGRF) for sequencing. Two hundred L of Promega membrane binding

solution was added to each tube of product. The tubes were spun at 14,000 rpm for 1 minute

and the flow‐through was discarded. Five hundred L of membrane was solution (from the

same kit as the membrane binding solution) was added and the tubes were spun at 14,000

rpm for 1 minute and the flow‐through was discarded. The above steps were repeated (with

addition of 500 L of membrane wash solution). The tubes were spun at 14,000 rpm as

empty and then the basket was transferred with membrane to a tube (Eppendorf 1.5), 50 L

of nuclease‐free water was added (from Promega kit mentioned above), and the tubes were

spun at 14,000 rpm for 1 minute. Finally, 6 L of RT‐PCR product was transferred to a 1.5

Eppendorf tube and 1 L of forward primer and 5 L of ultra pure distilled water was

added. The samples were sequenced by the AGRF Sanger routine sequencing service using

capillary separation on an AB 3730xl Sequencer.


51

3 results

The purpose of this project was to determine the presence/absence of GABA gene expression

in samples obtained from human sclera, choroid, RPE and retina. The results from the IHC

component of this project were inconclusive. The results from the RT‐PCR revealed the

presence of mRNA representing GABAA and GABAC receptor subunits throughout all ocular

layers tested, including the cells grown ex vivo. In addition, the cultured scleral cells and one

of the retinal donors yielded positive results for GABAC. Due to unresolvable issues with

contamination, we were not able to obtain consistent results for the housekeeping gene,

GAPDH, or any other housekeeping genes attempted. We observed positive results

representing the housekeeping control but also positive results in the case of the RT‐ve

control (refer to Table A8 in Appendix 3). We believe housekeeping gene contamination

may have been present in a communal reagent used during RNA extraction. Cleaned RT‐

PCR product representing each positive result was sent to the AGRF; our results were

verified. FC results revealed cell markers consistent with mesenchymal stromal cells.

3.1 Immunohistochemistry: Experiment #1

Shown in Figure 3.1 and Figure 3.2 is a representative example of the IHC (SMA – smooth

muscle actin – 1:100 and GABAA Alpha 1 1:100) undertaken during this project using donor

6397L (other donors used: 6806R & 6813R). IHC using all four antibodies was performed on

all four regions of the sclera using donor 6397L; no significant difference in staining for each

region was observed. Therefore, the data shown only concerns one region – the inferior

region – as a representative sample. A negative control was also performed by omission of

the primary antibody step. Due to concerns that bleaching may have reduced antigen‐

antibody interactions (Momose et al., 2011), the problem of an increased basophilic tissue,

and inconclusive results from the IHC staining, it was decided to employ molecular

techniques for future work.


52

Figure 3.1: IHC – SMA – performed on donor 6397L (sections taken from inferior region).

(A) Non‐bleached control showing neural retina, RPE, choroid and some scleral tissue; (B)

Non‐bleached SMA (1:100); (C) deeper view of (B); (D) Bleached control showing full width

of sclera. Bleaching, in combination with a blocking step, has resulted in tissue oxidation.

The tissue now appears washed‐out and the nuclei are pale; (E) Deeper view of (D); (F)

Bleached SMA (1:100) showing the cross‐section of a blood vessel.


53

Figure 3.2: IHC – GABAA Alpha 1 – performed on donor 6397L (sections taken from inferior

region).

(A) Non‐bleached control showing neural retina, RPE, choroid and some scleral tissue; (B)

Non‐bleached GABAA Alpha 1 (1:100); (C) Bleached GABAA Alpha 1 (1:100); (D) Non‐

bleached control showing deeper view of (A); (E) Non‐bleached GABAA Alpha 1 (1:100)

showing deeper view of (B); (F) Bleached GABAA Alpha 1 (1:100) showing deeper view of

(C); (G) Higher resolution of positive‐stained stromal cells; non‐bleached GABAA Alpha 1

(1:100); (H) Higher resolution of dendritic‐looking cells; non‐bleached GABAA Alpha 1

(1:100), (I) Some potential staining and some background staining; bleached GABAA Alpha 1

(1:100).

3.2 Flow Cytometry: Experiment #2

Crude cultures grown as fibroblast cells to passage 3 from the scleral explants – donors

6952L and 6953L – were characterised using FC and returned the following results for cell

surface markers: 99% positive for CD90, CD73, and CD105 and 15‐20% CD45 population.

From these results it could be deduced that the mesenchymal stromal cell population was at


54

about 80‐85% (Bray et al., 2012, Dominici et al., 2006). The cells were also 98% positive for

CD44 and negative results were obtained for CD31 & CD34. Any results using GABA

antibodies were inconclusive. A graph depicting the FC data (Figure 3.3) and the raw data

(Table 3.1) are shown on the following page.


55

Figure 3.3: Shown above is the percentage of live scleral‐derived stromal cells (passage 3). Ninety‐eight percent of the cells were positive for CD44

and 99% of the cells were positive for CD90, CD73, and CD105 and 15‐20% negative for CD45; based upon the expression of these cell surface

antigens, it was determined that the cell cultures were populated with 80‐85% mesenchymal stromal cells. Antibodies against GABAA‐α1, GABAAB‐

2, GABR‐R1, and GABR‐R2 were also utilised, as shown in the key for the table, but no positive results were obtained.

Table 3.1: Raw data analysis obtained from FC for human scleral stromal cells.

Antibody CD31 CD34 CD44 CD45 CD73 CD90 CD105 GABAA‐α1 GABAAB‐2 GABR‐R1 GABR‐R2

1.06 1.97 98.7 20.6 99.3 94.7 99.3 0.11 0 0.00346 0

0.84 2.33 98.9 19.4 99.5 93.9 99.4 0.055 0.00219 0 0.00354

0.14 1.61 99.8 17.4 99.6 94.8 99.1 0.093 0.00416 0.00349 0

19 98.8 97.9 98.3 0.014 0.02 0.00407

0.27 0.4 98.4 15.9 99.6 98.6 99 0.014 0.01 0

0.18 0.26 98.6 15.7 99.3 98.5 98.9 0.01 0.00512 0.015

Mean 0.498 1.314 98.88 18 99.35 96.4 99 0.086 0.007392 0.007012 0.003768

SD 0.37791 0.934949 0.544977 1.98897 0.275379 2.154066 0.389872 0.02816 0.006104 0.006524 0.005812


56

3.3 Analysis of GABA Receptor Gene Expression in Ocular Tissues and Cells: Experiment #3

3.3.1 RT‐PCR Amplification of GABA Receptors

Expression of the genes for several GABA receptor subunits were found in all layers of the

eye – sclera, choroid/RPE, and retina – and the mesenchymal stromal cells. Refer to Table 3.2

for the results and levels of amplification and Appendix 3 for pictures of all gels. Donors

6952 and 6953 presented positive results for GABAA Alpha 4 v1‐3 in all layers of the eye and

the mesenchymal stromal cells. Other genes observed throughout various layers and cells

were GABAA Beta 2 v1 & 2, GABAA Gamma 1, GABAA Gamma 2 v1‐3, GABAC Rho 1, and

GABAC Rho 2.

The molecular results from the three scleral donors revealed the presence of multiple GABA

receptor subtypes. All three donors demonstrated positive results for GABAA Beta 2 v1 & 2,

GABAA Gamma 1, GABAA Gamma 2 v1‐3, GABAC Rho 1, and GABAC Rho 2. Donor 6952

also produced positive results for GABAA Alpha 4 v1‐3 and GABAA Beta 3 v1‐4. Donor 6953

yielded the same results as Donor 6952. Donor 6954 provided positive results for GABAA

Alpha 6 and GABAA Pi as well as the five subtypes mentioned above.

The most variety in the expression of GABA receptor subunits was observed for the scleral‐

derived mesenchymal stromal cells. Donor 6952 yielded positive results for 16 subunits out

of a possible 21 and donor 6953 produced positive results for 13. Both tested positive for

GABAA Alpha 1 v1‐7, GABAA Alpha 4 v1‐3, GABAA Beta 1, GABAA Beta 2 v1 & 2, GABAA

Beta 3 v1‐4, GABAA Gamma 1, GABAA Gamma 2 v1‐3, GABAA Epsilon, GABAA Theta,

GABAA Pi, GABAB R1, and GABAC Rho 2. Donor 6952 also tested positive for GABAA Alpha

5 v1 & 2, GABAA Gamma 3, GABAB R2, and GABAC Rho 1. In addition to the shared

subunits with donor 6952, donor 6953 tested positive for GABAA Alpha 6.

The only GABA receptor subunit present in choroid/RPE tissues taken from donor 6952

appeared to be GABAA Alpha 4 v1‐3. GABAA Alpha 4 v1‐3 was also present in 6953; other

subtypes expressed in this donor included GABAA Beta 2 v1 & 2, GABAA Gamma 1, GABAA

Pi, GABAC Rho 1, and GABAC Rho 2.

In terms of the retinal analysis, both donors yielded positive results for GABAA Alpha 4 v1‐3,

GABAA Beta 2 v1 & 2, GABAA Gamma 2 v1‐3, GABAA Pi, GABAC Rho 1, and GABAC Rho 2.


57

In addition, donor 6953 tested positive for GABAA Gamma 1, GABAA Gamma 3, and GABAB

R2.

We experienced unresolvable issues with contamination of housekeeping genes despite

replacing all stock used during reverse‐transcription and RT‐PCR, trialling communal

housekeeping gene primer stock – such as beta‐2‐microglobulin, 18s rRNA, and PBGD, and

reordering another batch of GAPDH forward and reverse primers.

To confirm that the RT‐PCR products amplified using the specific primer pairs were in fact

the target gene, RT‐PCR products corresponding to each individual amplification reaction

were sequenced. This confirmed the specificity of the RT‐PCR products to their intended

gene target sequence and validates the results of the RT‐PCR GABA gene expression profiles

determined for each of the donor tissue and cell samples. The sequences, and their matched

sequence from the Genbank database, are shown in Appendix 4.


58

Table 3.2: The results for each of the GABA receptor subunit genes as demonstrated by RT‐PCR. Refer below for a key showing the colour shades

representing the level of amplification of each gene.

Tissue/Cells: Donor GABA A

ALPHA 1 V1‐V7

GABA A

ALPHA 2 V1 & V2

GABA A

ALPHA 3

GABA A

ALPHA 4 V1‐V3

GABA A

ALPHA 5 V1 & V2

GABA A

ALPHA 6

GABA A

BETA 1

GABA A

BETA 2 V1 & V2

GABA A

BETA 3 V1‐V4

GABA A

GAMMA 1

Sclera: 6952

Sclera: 6953

Sclera: 6954

Mesenchymal Stromal Cells: 6952


Choroid/RPE: 6952

Choroid/RPE: 6953

Retina: 6952

Retina: 6953

Weak Intermediate Strong


59

Tissue/Cells: Donor GABA A

GAMMA 2 V1‐V3

GABA A

GAMMA 3

GABA A

EPSILON

GABA A

DELTA

GABA A

THETA

GABA A

PI

GABA B

R1

GABA B

R2

GABA

RHO 1

GABA

RHO 2

GABA

RHO 3

Sclera: 6952

Sclera: 6953

Sclera: 6954



Choroid/RPE: 6952

Choroid/RPE: 6953

Retina: 6952

Retina: 6953

Weak Intermediate Strong


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4 discussion

4.1 Summary of Findings

The major question to be answered by this research project was: “Are GABA receptors

present in human scleral tissue?” We predicted there would be GABA receptors present,

most likely GABAC Rho 1, based on a recent animal study which found GABAC Rho 1

present in chick sclera (Cheng et al., 2011); note: other receptor subtypes were not studied.

The three scleral donors yielded positive gene expression results for GABAA and GABAC

receptor subunits using RT‐PCR. Of interest to this project was the fact that GABA

receptors, including GABAA Alpha 1 and GABAC Rho 1, have been found to be present in the

human retina (Davanger et al., 1991, Crooks and Kolb, 1992, Vardi and Sterling, 1994, Gussin

et al., 2011). As we collected the scleral tissue in the form of eye cups and therefore had

access to the tissue, we also investigated the presence of GABA receptors in the choroid/RPE

and retina, as well. The choroid/RPE and retinal samples yielded positive results for GABAA

and GABAC and one of the retinal donors was positive for GABAB. In addition to the above,

human scleral cells were cultured and tested for the presence of GABA receptors. The scleral

cells were positive for GABAA, GABAB, and GABAC. Immunophenotyping of these cultures

indicated a cell surface expression profile similar to that displayed by mesenchymal stromal

cells grown from other human tissue, including the corneal limbus (Bray et al., 2012). It is

currently known (based on evidence from animal studies) that the inhibitory

neurotransmitter GABA influences eye growth and, accordingly, refractive development

(Stone et al., 2003, Leung et al., 2005, Chebib et al., 2009b). Therefore, the discovery of GABA

receptor mRNA, mainly GABAA and GABAC, in different layers of the human eye has

important implications for future research in the field of refractive errors.

4.2 Limitations of Immunohistochemistry

The IHC component of the project revealed good evidence of GABA receptor expression in

one donor; however, this could not be repeated in two other donors and the requirement for

melanin bleach further complicated the clarity of results. Although the results for IHC were

variable, it is important to note that the positive results for the SMA antibody might suggest

the lack of immunoreactivity is associated only with the GABA antibody‐antigen interactions

(in the case that there is expression and that the lack of immunoreactivity is not simply a lack


61

of expression). A possible explanation for the variable GABA results could be related to

uncontrollable differences in the time between donor death and when the tissue was

retrieved and/or ultimately placed in fixative. Another variable is due to the time the tissue

spends in storage before being fixed and, in turn, the quality of the tissue. Furthermore,

better results may have been obtained if frozen eye sections were available – not just

formalin‐fixed – or if the IHC had been performed on cultured scleral cells. Formalin

fixation cross‐links proteins and can hinder the antibodies from gaining entry to binding

sites. However, despite the fact that frozen eye sections would circumvent this problem, the

morphology of the tissue is generally not as high quality. We would recommend future

studies utilise epitope, or antigen, retrieval techniques that would reduce cross‐linking and

improve binding of antibodies. Further studies utilising a larger number of tissue samples

and different staining techniques (e.g., use of different immunoperoxidase chromagen not

requiring melanin bleach, such as Fast Red) should hopefully resolve this issue. Given the

present time restrictions, however, it was deemed necessary to shift the focus of the project

from a histological approach to a gene expression approach.

4.3 Polymerase Chain Reaction Methodological Issues

Utilising RT‐PCR, all 21 genes for the GABA receptor subunits were tested in a minimum of

two donors (n=3 for scleral tissue). GABA gene expression was found to be present in all

layers of the eye – sclera, choroid/RPE, and retina – and was found to be present in cultured

scleral‐derived mesenchymal stromal cells, as well. Whilst the RT‐PCR component yielded

results and the IHC component proved variable, it is important to note that it is easier to

optimise an RT‐PCR method as the primers are designed to be very specific to the target.

IHC is reliant on interactions between proteins – the antibody, the target, and the

environment of the experiment; salt, pH, temperature, and other factors can affect the

affinity of the antibody for the GABA receptor protein in the tissue. In addition, the

processing of the tissue itself can sometimes mask, or hide, the epitopes that the antibody

recognises.

4.4 GABA Receptors in Sclera

GABAA (5/16 possible GABAA receptor subunits for all three donors) and GABAC (2/3

GABAC receptor subunits for all three donors) receptor subunits were found to be present in

the three scleral donors. Donors 6952, 6953, and 6954 returned positive results for GABAA


62

and GABAC receptor subunits (no positive results were observed in any donor for GABAB

receptor subunits). To our knowledge, no one has demonstrated the presence of GABA

receptors in scleral tissue excepting one study demonstrating the presence of GABAC Rho 1

receptors in chick sclera (Cheng et al., 2011). As mentioned previously, another study found

expression of GABARAP, a gene associated with GABAA, in the human sclera (Young et al.,

2003). The majority of GABA receptor subunits were observed in the scleral‐derived

mesenchymal stromal cells. Between the two donors, only four out of the twenty‐one GABA

receptor subunits analysed were not expressed.

4.5 GABA Receptors in Retinal Pigment Epithelium & Choroid

Of particular note to this project, GABAA and GABAB receptor signalling pathways were

recently discovered in the RPE (Booij et al., 2010). The GABAA finding is consistent with our

results for the choroid/RPE samples obtained from both donors analysed as receptor

subunit(s) for GABAA were found to be present. It has been previously thought that the RPE

may have a role in clearing GABA from the subretinal space (species analysed: Rana

Catesbeiana) (Peterson and Miller, 1995). Similarly, Booij et al., 2010 have also suggested that

the GABA receptor signalling pathway in human RPE may function to uptake GABA from

the subretinal space (Booij et al., 2010). We did not find GABAB present in choroid/RPE from

either donor.

4.6 GABA Receptors in Retina

We noted the presence of several GABA receptor subunits in both donors. Some of our

results – GABAC Rho 1 and GABAC Rho 2 – from both retinal donors were consistent with

published literature (Gussin et al., 2011, Hackam et al., 1997, Wang et al., 1994) and one

previous finding (Vardi and Sterling, 1994) – GABAA Alpha 1 – was not observed in this

study. In addition, previously unpublished positive results – GABAA Alpha 4 v1‐3, GABAA

Beta 2 v1 & 2, GABAA Gamma 1, GABAA Gamma 2 v1‐3, GABAA Gamma 3, GABAA Pi, and

GABAB R2 – for both donors were also observed. Two studies (Gussin et al., 2011, Hackam

et al., 1997) have demonstrated positive findings for GABAC Rho 1 in human retinal tissue;

we also found this receptor subtype present in both of our donors. GABAC Rho 2 has been

shown previously to be present in human retinal tissue (Hackam et al., 1997, Wang et al.,

1994); we observed positive results for this receptor subtype in our donors. However, a

previous study (Vardi and Sterling, 1994) also reported a positive result for the GABAA


63

Alpha 1 receptor subtype in the plexiform layers and many amacrine and ganglion cell

somas in the inner nuclear layer and ganglion cell layer using tissue obtained from human

retina. We did not observe positive results for this receptor subtype in either of our two

donors (as product for GABAA Alpha 1 was found to be expressed in the mesenchymal

stromal cells, it would be assumed that the primer worked and the gene was not expressed

in the retinal samples). As well as the positive results for the two receptor subtypes

mentioned above – GABAC Rho 1 and GABAC Rho 2 – we also observed positive findings for

GABAA Alpha 4 v1‐3, GABAA Beta 2 v1 & 2, GABAA Gamma 2 v1‐3, and GABAA Pi for both

donors. Donor 6953 also demonstrated positive results for GABAA Gamma 1, GABAA

Gamma 3, and GABAB R2.

4.7 Limitations of Flow Cytometry

The FC results using GABA antibodies were inconclusive. The antibodies used for FC

correlated with positive results from the RT‐PCR for both donors; the only exception was for

the receptor GABAB R2 in donor 6953. Inconclusive, perhaps negative, results may have

been due to the processes involved in FC; if the receptors are present on the surface of the

cell, the cell surface proteins may have been cleaved and, in the process, the GABA receptors

were lost. For example, as we used trypsin during the course of FC, we may have degraded

surface antigens (and possibly intracellular antigens). Another possibility is that the

receptors are present internally and FC is not the best method to characterise these receptors.

Furthermore, a lack of results for FC may be due to cell culturing conditions not being ideal

for preserving the GABA receptors.

4.8 Relevance to Myopia

As mentioned previously, GABAC‐selective antagonists have proven to be more potent than

either GABAA‐ or GABAB‐selective antagonists in inhibiting ocular growth (Stone et al., 2003,

Chebib et al., 2009a, Chebib et al., 2009b). Our finding is of significant interest to these

previous studies as we found GABAC to be present in each of the layers – sclera,

choroid/RPE, and retina (excluding only the choroid/RPE layer of donor 6952) – of both

donors’ eyes, as well as being present in the mesenchymal stromal cells. Also of

pharmacological interest is the fact that topirimate, an anticonvulsant and GABAA agonist,

has been associated with myopia as a notable side effect (Zilliox and Russell, 2011, Craig et

al., 2004, Verrotti et al., 2007, Bhattacharyya and Basu, 2005).


64

4.9 Limitations

Perhaps the most obvious limitation of this study was the use of cadaveric tissue; the study

was limited to studying a post‐mortem endpoint rather than being able to examine the sclera

(and other tissues) during the course of onset and development of myopia – unlike animal

studies. It must be noted that one major limitation of animal studies are that form‐

deprivation myopia and lens‐induced myopia may involve different mechanisms to those

involved in the myopia onset and development observed in humans. However, animal

studies are very useful as they can inform and thus guide human‐based work. In many

instances, pathways are conserved across species. It is clear that there are pros and cons to

both approaches of research.

As part of the post‐mortem processes, the tissue must undergo serological testing before it

can be released. The delay for results can be as long as two to three days and, during this

period of time, tissue, and definitely the RNA within the tissue, can be subject to degrading

and different storage conditions. A housekeeping gene – GAPDH – was used to confirm the

integrity of the RNA extracted from the tissues that was then used to make the cDNA

template for the RT‐PCR amplification of the GABA receptor sequences. We observed

strong expression of the housekeeping gene and, therefore, in accordance with the high RNA

yields observed, are confident that the tissue used was of a suitable quality.

As the donors ranged in ages from 61‐86 years, it is possible that the age of the donors

impacted on the quality of the tissue. Therefore, it is possible that different results may be

obtained from younger eyes. We would only be able to address this potential confounding

variable through future research.

As mentioned previously, we were not provided information about the donors’ eyes in terms

of the presence of ocular pathologies or refractive error. We can only speculate that the

presence of either a diseased state or refractive error may potentially influence the results. It

would be interesting to compare results obtained from emmetropic and myopic individuals

in future studies, if possible.

It would be recommended that future studies use a larger sample size. For this project, we

used two donors for choroidal/RPE, retinal and stromal cell samples; this simply served as a


65

snapshot and provides preliminary data for future avenues of research.

4.10 Real World Implications

At a practical level, the discovery of GABAC receptor mRNA throughout all layers of the eye,

including the scleral‐derived mesenchymal stromal cells, is of particular relevance to

pharmacological studies that have demonstrated that GABAC receptor antagonists can

inhibit ocular growth. With the knowledge that mRNA for these receptors is present, it

supports the hypothesis that GABAC receptors are pharmacological targets in human sclera

for agents that may potentially inhibit, or at least slow, the progression of myopia based on

results from animal studies.

4.11 Future Directions

There are several questions – both from histological and gene expression perspectives –

arising from the current project. Firstly, it would be important to follow‐up the current

project with a protein detection and quantification technique, such as Western blot and

quantitative RT‐PCR. As we have deduced the presence of mRNA for targeted GABA

receptor subtypes, Western blot would allow detection of GABA receptor protein, if present.

We did not undertake quantification studies for this project for a few reasons. This was

preliminary work and therefore we were most interested in presence/absence. The RNA was

not of the best quality and so there was no real justification for real‐time PCR. However, this

study does justify collection of eye tissue under more appropriate conditions for RNA

extraction and, under those circumstances, a qualitative study would be worthwhile. It may

even be as simple as placing the eye in RNA Later Solution or even more ideal, dissecting the

tissue on immediate removal from the patient and snap freezing in liquid nitrogen.

The next big overarching questions to address would be: If GABA protein is present, what

could the functional purpose(s) of these receptors be and what is their localisation? GABA

receptors are known to play a key inhibitory role in the CNS and may be pharmacologically

targeted for improved outcomes for individuals with myopia; specific, key role(s) in the eye

itself are yet to be deduced. Given a lack of data regarding the significance of the expression

of the different receptor subtypes, interpretation is currently difficult and this is something

that could be addressed in future research. If the protein for these GABA receptors is indeed

present, it would be interesting to localise where the receptors are within the individual


66

layers of the eye. Follow‐up work would include IHC and immunocytochemistry using

relevant antibodies. As mentioned above, given the similar colour of melanin granules

compared with IHC staining using DAB and the complications of melanin bleach, I would,

in future, elect to use a different IHC system incorporating use of the chromagen Fast Red,

which can be distinguished from melanin.

Another possible avenue of future research would be to determine whether the receptors are

up‐regulated or down‐regulated in myopic eyes as compared with emmetropic eyes. mRNA

for the receptors is present but a next question to address would be whether they are

functional or not and, if so, what their biological contribution to myopia might be. One way

this could be undertaken would be through determining the influence of GABA drugs on the

different tissues and cells in terms of morphology, proliferation, expression of ECM‐

remodelling enzymes, and production of ECM components. Furthermore, it would be

interesting to look into possible growth factors that may be affected by or regulated in the

presence of GABAergic receptor modulators, as recommended previously (Leung et al.,

2005).

Furthermore, it would be useful to deduce where a supply of GABA active substance, or

GABA neurotransmitter, would come from. As the retina contains GABAergic neurons, we

hypothesise that active substance would most likely be supplied from the retina to the other

layers of the eye, including the sclera. It is also important to note, however, that GABA has

been shown to be present in cerebral blood vessels; both GAD, the enzyme for GABA

synthesis, and GABA‐T, the catabolic enzyme for GABA, occur in cerebral blood vessels

(Imai et al., 1991). More specifically, 25‐50% of the GABA present in whole blood is found

within the plasma fraction and the rest is localised in formed elements (Ferkany et al., 1979).

Therefore, an active supply may be present in the choroidal circulation. Finally, it would be

interesting to determine if these GABA receptors are involved in other ocular functions,

aside from potential involvement in eye growth regulation.

Finally, it would be necessary to consider what the implication(s) would be for a therapeutic

aimed at minimising proprioceptive control of scleral growth and the potential impact on

IOP. One rat study (Samuels et al., 2012) found that the use of GABAA receptor antagonist,

bicuculline, microinjected into the dorsomedial and perfornical hypothalamus evoked a


67

substantial increase in IOP. It would be important to undertake further pharmacological

research in this area.

4.12 Conclusion

In summary, it appears that expression of GABA receptors – mainly GABAA and GABAC – is

present, based on positive mRNA findings, throughout the different layers of the eye,

including the scleral layer and scleral‐derived mesenchymal stromal cells. The finding of

GABAC receptor mRNA is of particular note based on previous pharmacological studies

using animals that found that GABAC antagonists are capable of inhibiting ocular growth.

This study has provided novel, preliminary gene expression data that may contribute to an

understanding of the myopia signalling cascade and further exploration of the contribution

of GABA receptors in these tissues will, in due course, hopefully lead to effective

preventative strategies for myopic onset or treatments aimed at slowing (or inhibiting)

progression.


68

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Appendix one: ANIMAL STUDIES – background

Animal models have provided invaluable insights into some of the processes occurring

during the onset and progression of myopia. The development of animal models of myopia

occurred when Wiesel and Raviola discovered that suturing the eyes of monkeys induced

myopia (Wiesel and Raviola, 1977). In the intervening years, experiments using techniques,

such as form‐deprivation (Wiesel and Raviola, 1977) and optical defocus (Schaeffel et al.,

1988), have demonstrated the role of vision in the regulation (and alteration) of eye growth

(Collins et al., 2006). [However, it must be noted that it is still unclear how correlative

induced myopia in animals may be to physiologic myopia in humans (Leo and Young,

2011).] Refer to Figure A1 for a diagrammatic representation of the processes occurring

during form‐deprivation and myopic defocus in animal studies.

Figure A1: Animal models depicting the processes involved during induced‐hyperopia

myopic defocus (portrayed on the left) and induced‐myopia hyperopic defocus (portrayed

on the right) (Rymer and Wildsoet, 2005).

Several animal models have been used to date (refer to Table A1); the most common model is

the chick model (Metlapally and McBrien, 2008). While chick models, in particular, have

allowed greater understanding of myopia, there are some fundamental and important

differences – in comparison to mammalian eyes – in both the structure of the eye and in the

way the eye changes in the context of myopia (Morgan, 2003). For example, chick sclera is

bi‐layered: it consists largely of cartilaginous tissue but contains a small layer of fibrous


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tissue (Morgan, 2003). The human eye consists of a single fibrous layer only (Summers Rada

et al., 2006). In most animals, the sclera is supported with cartilage and even bone; is

thought that the less rigid fibrous structure of the human sclera allows a more even

distribution of the blood supply to the choroid, and hence the retina, during large rotations

of the eyeball (Watson and Young, 2004). Another notable feature of the chick eye ‐ absent in

the human eye – is the presence of ossicles, or rings of bony structures, located near the

corneal limbus (Wallman and Winawer, 2004).

Table A1: Recent examples of animal models used for myopia research [for an extensive list

of original papers representing studies for each of these species, refer to (Barathi et al., 2008)].

Species References

Avian: Gallus gallus domesticus (Chen et al., 2010, Summers Rada et al., 2006, Wallman et al., 1978)

Cat (Jinren and Smith III, 1989, Gollender et al., 1979)

Fish: Oreochromis niloticus (Shen et al., 2005)

Guinea Pig: Cavia porcellus (Howlett and McFadden, 2002)

Marmoset: Callithrix jacchus (Troilo and Judge, 1993, Troilo et al., 2006, Troilo et al., 2007)

Monkey: Macaca mulatta (Wiesel and Raviola, 1977, Summers Rada et al., 2006)

Mouse: Mus musculus (Barathi and Beuerman, 2011, Barathi et al., 2008)

Squirrel: Sciurus carolinensis (McBrien et al., 1993)

Tree Shrew: Tupaia glis belangeri (Abbott et al., 2011, Sherman et al., 1977, Summers Rada et al., 2006)


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Appendix Two: Gross Anatomy of Human Sclera

Following are a series of figures (Figures A2 – A7) outlining the relative positions of the

major muscles and blood vessels surrounding the human eye. These landmarks were used

to orient the regions of interest – superior, inferior, medial, and lateral – of the eyes used for

the immunohistochemical component of this project.

Figure A2: Some of the major muscles surrounding the eyeball; shown are the relative

positions of the insertions of the horizontal and vertical rectus muscles (Maza et al., 2012).

Figure A3: The insertions of the superior oblique and inferior oblique muscles (Maza et al.,

2012).


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Figure A4: Vascular supply to the globe, highlighting the relationships between the internal

carotid, ophthalmic, central retina, long posterior ciliary, short posterior ciliary, anterior

ciliary, and lacrimal arteries (Maza et al., 2012).

Figure A5: Sites of perforation of the sclera by the long and short posterior ciliary arteries,

the short posterior ciliary veins, and the inferior and superior vortex veins (Maza et al.,

2012).


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Figure A6: The relationships between the lacrimal, vortex, short posterior ciliary, inferior

ophthalmic, retinal, supraorbital, and superior ophthalmic veins (Maza et al., 2012).

Figure A7: The relationships, shown as a transverse section of the globe, between the short

posterior ciliary, long posterior ciliary, and anterior ciliary arteries and short posterior

ciliary, anterior ciliary, and vortex veins (Maza et al., 2012).


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Appendix Three: Gel results

The following tables provide all results for polymerase chain reaction undertaken during this

project. We observed results consistent with intron genomic DNA and splice variants, for

example. We have included maps in Appendix 5 that outlines the positions of the primers

with respect to introns and exons.

Table A2: RT‐PCR results for mesenchymal stromal cells obtained from donor 6952. The aim

of this study was to determine the presence or absence of GABA receptors in human scleral

tissue and cultured human scleral cells, as well as choroidal/RPE and retinal tissue.

Scleral Stromal Cells – 6952 – 65°C (Pages 87‐88 of Laboratory Notebook)

Lane: Top Row Primer Notes

1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7

3 Primer #2: GABA A Alpha 2v1&2

4 Primer #3: GABA A Alpha 3

5 Primer #4: GABA A Alpha 4v1‐3 Intermediate expression


7 Primer #6: GABA A Alpha 6 Intermediate expressiona

8 Primer #7: GABA A Beta 1

9 Primer #8: GABA A Beta 2v1&2 Weak expression

10 Primer #9: GABA A Beta 3v1‐4 Strong expression

11 Primer #10: GABA A Gamma 1

12 Primer #11: GABA A Gamma 2v1‐3



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14 Primer #13: GABA A Epsilon Presence of faint band

15 Primer #14: GABA A Delta

16 Primer #15: GABA A Theta

17 Primer #16: GABA A Pi

18 Primer #17: GABA B R1 Weak expression

19 Primer #18: GABA B R2 Weak expression

20 Primer #19: GABA C Rho 1 Weak expression

21 Primer # 20: GABA C Rho 2

22 Primer #21: GABA C Rho 3

Lane: Bottom Row Primer Notes

1 Marker VIII

2 Housekeeping control Housekeeping gene: GAPDH – No expression

3 RT‐ve control Housekeeping gene: GAPDH

4 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control

5 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control

6 Primer #3: GABA A Alpha 3 PCR‐ve control




10 Primer #7: GABA A Beta 1 PCR‐ve control

11 Primer #8: GABA A Beta 2v1&2 PCR‐ve control a As the band appears too high based on the predicted base pair, we suspect that this is caused by intron genomic

DNA. The primer worked but the gene was not present and, therefore, the primer serves as an internal control.


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1 Marker VIII






7 Primer #6: GABA A Alpha 6 Strong expression

8 No load

9 Primer #7: GABA A Beta 1

10 Primer #8: GABA A Beta 2v1&2 Weak expression


12 Primer #10: GABA A Gamma 1 Presence of faint band

13 Primer #11: GABA A Gamma 2v1‐3


15 Primer #13: GABA A Epsilon



18 Primer #16: GABA A Pi Weak expressiona

19 Primer #17: GABA B R1



22 Primer # 20: GABA C Rho 2 Weak expressiona



1 Marker VIII





98





9 Primer #8: GABA A Beta 2v1&2 PCR‐ve control


11 Housekeeping control GAPDH – Strong expression a As the bands appear too low for the predicted base pair, it is possible that this is caused by a splice variant.


99




Scleral Stromal Cells – 6952 & 6953 – 63°C (Pages 91‐92 of Laboratory Notebook)


1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 6952

3 Primer #2: GABA A Alpha 2v1&2 6952

4 Primer #3: GABA A Alpha 3 6952

5 Primer #5: GABA A Alpha 5v1&2 6952 – Weak expression

6 Primer #7: GABA A Beta 1 6952

7 Primer #8: GABA A Beta 2v1&2 6952 – Strong expression

8 Primer #10: GABA A Gamma 1 6952 – Weak expression

9 Primer #11: GABA A Gamma 2v1‐3 6952 – Strong expression

10 Primer #12: GABA A Gamma 3 6952

11 Primer #13: GABA A Epsilon 6952 – Intermediate expression

12 Primer #14: GABA A Delta 6952

13 Primer #15: GABA A Theta 6952

14 Primer #16: GABA A Pi 6953 – Strong expression

15 Primer #17: GABA B R1 6952 – Weak expression

16 Primer #18: GABA B R2 6952 – Intermediate expression

17 Primer #19: GABA C Rho 1 6952 – Intermediate expression

18 Primer # 20: GABA C Rho 2 6953 – Strong expression

19 Primer #21: GABA C Rho 3 6952


1 Marker VIII All of the following: 6952

2 Housekeeping control GAPDH – Strong expression




100





9 Primer #11: GABA A Gamma 2v1‐3 PCR‐ve control

10 Primer #12: GABA A Gamma 3 PCR‐ve control

11 Primer #14: GABA A Delta PCR‐ve control


101

Table A5: RT‐PCR results for mesenchymal stromal cells obtained from donors 6952 & 6953.

The aim of this study was to determine the presence or absence of GABA receptors in human

scleral tissue and cultured human scleral cells, as well as choroidal/RPE and retinal tissue.

Scleral Stromal Cells – 6952 & 6953 – 67°C (Pages 94‐97 of Laboratory Notebook)


1 Marker VIII






7 Primer #10: GABA A Gamma 1 6952 – Strong expression




11 Primer #17: GABA B R1 6952 – Strong expression







18 Primer #10: GABA A Gamma 1 6953 – Strong expression




22 Primer #17: GABA B R1 6953



1 Marker VIII

2 Housekeeping control (6952) GAPDH – Strong expression

3 RT‐ve control (6952) Housekeeping gene: GAPDH

4 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control (6952)


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5 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control (6952)

6 Primer #3: GABA A Alpha 3 PCR‐ve control (6952)

7 Primer #5: GABA A Alpha 5v1&2 PCR‐ve control (6953)

8 Primer #7: GABA A Beta 1 PCR‐ve control (6953)

9 Primer #10: GABA A Gamma 1 PCR‐ve control (6953)


103






1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 Weak expression




6 Primer #7: GABA A Beta 1 Weak expression



10 Primer #15: GABA A Theta Weak expression



1 Marker VIII

2 Housekeeping control Housekeeping gene: GAPDH – Strong expression









104

Table A7: RT‐PCR results for choroid/RPE, retina, and mesenchymal stromal cells obtained

from donors 6952 & 6953. The aim of this study was to determine the presence or absence of

GABA receptors in human scleral tissue and cultured human scleral cells, as well as


Choroid/RPE, Retina, Scleral Stromal Cells – 6952 & 6953 – 67°C (Pages 104‐105 of Laboratory Notebook)


1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 Choroid/RPE – 6952

3 Primer #2: GABA A Alpha 2v1&2 Choroid/RPE – 6952

4 Primer #10: GABA A Gamma 1 Choroid/RPE – 6952


6 Primer #14: GABA A Delta Choroid/RPE – 6952

7 Primer #17: GABA B R1 Choroid/RPE – 6952

8 Primer #21: GABA C Rho 3 Choroid/RPE – 6952

9 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control (Choroid/RPE – 6952)

10 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control (Choroid/RPE – 6952)

11 Primer #10: GABA A Gamma 1 PCR‐ve control (Choroid/RPE – 6952)

12 Primer #1: GABA A Alpha 1v1‐7 RT‐ve control (Choroid/RPE – 6952)




16 Primer #10: GABA A Gamma 1 Choroid/RPE – 6953 – Strong expression





21 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control (Choroid/RPE – 6953)

22 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control (Choroid/RPE – 6953)

23 Primer #10: GABA A Gamma 1 PCR‐ve control (Choroid/RPE – 6953)

24 Primer #1: GABA A Alpha 1v1‐7 RT‐ve control (Choroid/RPE – 6953)


26 Primer #10: GABA A Gamma 1 Scleral Stromal Cells– 6952 – Strong expression

27 Primer #17: GABA B R1 Scleral Stromal Cells – 6952

28 Primer #10: GABA A Gamma 1 PCR‐ve control (Scleral Stromal Cells – 6953)

29 Primer #17: GABA B R1 PCR‐ve control (Scleral Stromal Cells – 6953)

30 Marker VIII


105


1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 Retina – 6952

3 Primer #2: GABA A Alpha 2v1&2 Retina – 6952

4 Primer #10: GABA A Gamma 1 Retina – 6952


6 Primer #14: GABA A Delta Retina – 6952

7 Primer #17: GABA B R1 Retina – 6952

8 Primer #21: GABA C Rho 3 Retina – 6952

9 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control (Retina – 6952)

10 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control (Retina – 6952)

11 Primer #10: GABA A Gamma 1 PCR‐ve control (Retina – 6952)

12 Primer #1: GABA A Alpha 1v1‐7 RT‐ve control (Retina – 6952)




16 Primer #10: GABA A Gamma 1 Retina – 6953 – Strong expression





21 Primer #1: GABA A Alpha 1v1‐7 PCR‐ve control (Retina – 6953)

22 Primer #2: GABA A Alpha 2v1&2 PCR‐ve control (Retina – 6953)

23 Primer #10: GABA A Gamma 1 PCR‐ve control (Retina – 6953)

24 Primer #1: GABA A Alpha 1v1‐7 RT‐ve control (Retina – 6953)


26 Primer #1: GABA A Alpha 1v1‐7 RT‐ve control (Stromal Cells – 6952)

27 Housekeeping control Housekeeping gene: GAPDH – No expression

30 Marker VIII


106

Table A8: RT‐PCR results for choroid/RPE, retina, sclera, and mesenchymal stromal cells

obtained from donors 6952 & 6953. The aim of this study was to determine the presence or

absence of GABA receptors in human scleral tissue and cultured human scleral cells, as well

as choroidal/RPE and retinal tissue.

Choroid/RPE, Retina, Sclera, Scleral Stromal Cells – 6952 & 6953 – 60/67°C (Pages 106‐107 of Notebook)


1 Marker VIII

2 GAPDH – Sclera – 6952 Housekeeping control – Expression



5 GAPDH – Choroid/RPE – 6952 Housekeeping control – Expression

6 GAPDH – Choroid/RPE – 6953 Housekeeping control – Expression

7 GAPDH – Retina – 6952 Housekeeping control – Expression

8 GAPDH – Retina – 6953 Housekeeping control – Expression

9 GAPDH – Scleral Stromal Cells – 6952 Housekeeping control – Expression

10 GAPDH – Scleral Stromal Cells – 6953 Housekeeping control – Expression

11 Marker VIII

13 Primer #10: GABA A Gamma 1 Cells – 6952 – Strong expression

14 Primer #17: GABA B R1 Cells – 6953 – Intermediate expression


1 Marker VIII

2 GAPDH – Sclera – 6952 PCR‐ve control



5 GAPDH – Choroid/RPE – 6952 PCR‐ve control – Expression

6 GAPDH – Choroid/RPE – 6953 PCR‐ve control

7 GAPDH – Retina – 6952 PCR‐ve control

8 GAPDH – Retina – 6953 PCR‐ve control

9 GAPDH – Scleral Stromal Cells – 6952 PCR‐ve control

10 GAPDH – Scleral Stromal Cells – 6953 PCR‐ve control


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13 GAPDH – Sclera – 6952 RT‐ve control – Expression



16 GAPDH – Choroid/RPE – 6952 RT‐ve control – Expression

17 GAPDH – Choroid/RPE – 6953 RT‐ve control – Expression

18 GAPDH – Retina – 6952 RT‐ve control – Expression

19 GAPDH – Retina – 6953 RT‐ve control – Expression

20 GAPDH – Scleral Stromal Cells – 6952 RT‐ve control – Expression

21 GAPDH – Scleral Stromal Cells – 6953 RT‐ve control – Expression

22 Marker VIII


108

Table A9: RT‐PCR results for sclera obtained from donor 6952. The aim of this study was to

determine the presence or absence of GABA receptors in human scleral tissue and cultured

human scleral cells, as well as choroidal/RPE and retinal tissue.

Sclera – 6952 – 60/63°C (Pages 112‐113 of Laboratory Notebook)


1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 Sclera – 6952

3 Primer #3: GABA A Alpha 3 Sclera – 6952

4 Primer #5: GABA A Alpha 5v1&2 Sclera – 6952

5 Primer #7: GABA A Beta 1 Sclera – 6952

6 Primer #8: GABA A Beta 2v1&2 Sclera – 6952 – Strong expression

7 Primer #11: GABA A Gamma 2v1‐3 Sclera – 6952 – Intermediate expression

8 Primer #12: GABA A Gamma 3 Sclera – 6952

9 Primer #13: GABA A Epsilon Sclera – 6952

10 Primer #14: GABA A Delta Sclera – 6952

11 Primer #15: GABA A Theta Sclera – 6952

12 Primer #16: GABA A Pi Sclera – 6952

13 Primer #18: GABA B R2 Sclera – 6952

14 Primer #19: GABA C Rho 1 Sclera – 6952 – Weak expression

15 Primer # 20: GABA C Rho 2 Sclera – 6952 – Intermediate expression

16 Primer #21: GABA C Rho 3 Sclera – 6952









25 Marker VIII


109

Table A10: RT‐PCR results for sclera obtained from donor 6953. The aim of this study was

to determine the presence or absence of GABA receptors in human scleral tissue and

cultured human scleral cells, as well as choroidal/RPE and retinal tissue.



1 Marker VIII






7 Primer #11: GABA A Gamma 2v1‐3 Sclera – 6953 – Intermediate expression





12 Primer #16: GABA A Pi Sclera – 6953


14 Primer #19: GABA C Rho 1 Sclera – 6953 – Intermediate expression

15 Primer # 20: GABA C Rho 2 Sclera – 6953 – Strong expression










25 Marker VIII


110

Table A11: RT‐PCR results for sclera obtained from donor 6954. The aim of this study was





1 Marker VIII









10 Primer #11: GABA A Gamma 2v1‐3 Sclera – 6954 – Strong expression





15 Primer #16: GABA A Pi Sclera – 6954 – Intermediate expression


17 Primer #19: GABA C Rho 1 Sclera – 6954 – Intermediate expression

18 Primer # 20: GABA C Rho 2 Sclera – 6954 – Strong expression


30 Marker VIII


1 Marker VIII

2 GAPDH – Sclera – 6954 Housekeeping control

3 GAPDH – Sclera – 6952 RT‐ve control











30 Marker VIII


111

Table A12: RT‐PCR results for sclera obtained from donors 6952, 6953, & 6954. The aim of

this study was to determine the presence or absence of GABA receptors in human scleral


Sclera – 6952, 6953, & 6954 – 65°C (Pages 118‐119 of Laboratory Notebook)


1 Marker VIII




5 Primer #4: GABA A Alpha 4v1‐3 Sclera – 6952 – Strong expression



8 Primer #9: GABA A Beta 3v1‐4 Sclera – 6952 – Strong expression











19 Primer #4: GABA A Alpha 4v1‐3 Sclera – 6953 – Weak expression



22 Primer #9: GABA A Beta 3v1‐4 Sclera – 6953 – Strong expression








30 Marker VIII


1 Marker VIII


112






7 Primer #6: GABA A Alpha 6 Sclera – 6954 – Strong expression

8 Primer #9: GABA A Beta 3v1‐4 Sclera – 6954








30 Marker VIII


113

Table A13: RT‐PCR results for sclera obtained from donors 6952, 6953, & 6954. The aim of

this study was to determine the presence or absence of GABA receptors in human scleral


Sclera – 6952, 6953, & 6954 – 60/67°C (Pages 120‐121 of Laboratory Notebook)


1 Marker VIII



4 Primer #10: GABA A Gamma 1 Sclera – 6952 – Strong expression






10 GAPDH – Sclera – 6952 RT‐ve control – Contamination?



13 Primer #17: GABA B R1 PCR‐ve control



16 Primer #10: GABA A Gamma 1 Sclera – 6953 – Strong expression












28 Primer #10: GABA A Gamma 1 Sclera – 6954 – Intermediate expression


30 Marker VIII





114



5 GAPDH – Sclera – 6954 RT‐ve control – Contamination?




29 Marker VIII


115

Table A14: RT‐PCR results for choroid/RPE obtained from donor 6952. The aim of this study

was to determine the presence or absence of GABA receptors in human scleral tissue and


Choroid/RPE – 6952 – 60/63°C (Pages 122‐123 of Laboratory Notebook)


1 Marker VIII



4 Primer #3: GABA A Alpha 3 Choroid/RPE – 6952


6 Primer #7: GABA A Beta 1 Choroid/RPE – 6952

7 Primer #8: GABA A Beta 2v1&2 Choroid/RPE – 6952

8 Primer #11: GABA A Gamma 2v1‐3 Choroid/RPE – 6952


10 Primer #13: GABA A Epsilon Choroid/RPE – 6952


12 Primer #15: GABA A Theta Choroid/RPE – 6952

13 Primer #16: GABA A Pi Choroid/RPE – 6952



16 Primer # 20: GABA C Rho 2 Choroid/RPE – 6952


18 18s rRNA – Choroid/RPE – 6952 Housekeeping control – Expression

19 18s rRNA – Choroid/RPE – 6952 RT‐ve control




30 Marker VIII


116

Table A15: RT‐PCR results for choroid/RPE & retina obtained from donors 6952 & 6953. The

aim of this study was to determine the presence or absence of GABA receptors in human


Choroid/RPE & Retina – 6952 & 6953 – 65/67°C (Pages 124‐125 of Laboratory Notebook)


1 Marker VIII








9 B2M – Choroid/RPE – 6952 Housekeeping control – Expression

10 B2M – Choroid/RPE – 6952 RT‐ve control





15 Primer #10: GABA A Gamma 1 Choroid/RPE – 6953 – Intermediate expression





20 B2M – Choroid/RPE – 6953 Housekeeping control – Expression











117

30 Marker VIII


1 Marker VIII


3 B2M – Retina – 6952 Housekeeping control – Expression

4 B2M – Retina – 6952 RT‐ve control





9 Primer #10: GABA A Gamma 1 Retina – 6953 – Strong expression









30 Marker VIII


118

Table A16: RT‐PCR results for retina obtained from donor 6953. The aim of this study was



Retina – 6953 – 63/65°C (Pages 126‐127 of Laboratory Notebook)


1 Marker VIII



4 Primer #3: GABA A Alpha 3 Retina – 6953


6 Primer #7: GABA A Beta 1 Retina – 6953

7 Primer #8: GABA A Beta 2v1&2 Retina – 6953 – Strong expression

8 Primer #11: GABA A Gamma 2v1‐3 Retina – 6953 – Strong expression

9 Primer #12: GABA A Gamma 3 Retina – 6953 – Intermediate expression

10 Primer #13: GABA A Epsilon Retina – 6953


12 Primer #15: GABA A Theta Retina – 6953

13 Primer #16: GABA A Pi Retina – 6953 – Strong expression

14 Primer #18: GABA B R2 Retina – 6953 – Intermediate expression

15 Primer #19: GABA C Rho 1 Retina – 6953 – Strong expression

16 Primer # 20: GABA C Rho 2 Retina – 6953 – Strong expression







30 Marker VIII


119


obtained from donor 6952. The aim of this study was to determine the presence or absence

of GABA receptors in human scleral tissue and cultured human scleral cells, as well as


Retina, Choroid/RPE, Sclera, & Scleral Stromal Cells – 6952, 3 & 4 – 63/65°C (Pages 130‐131 of Notebook)


1 Marker VIII

2 GAPDH – Choroid/RPE – 6952 Housekeeping control

3 GAPDH – Choroid/RPE – 6953 Housekeeping control

4 GAPDH – Retina – 6952 Housekeeping control

5 GAPDH – Retina – 6953 Housekeeping control




9 GAPDH – Scleral Stromal Cells – 6952 Housekeeping control

10 GAPDH – Scleral Stromal Cells – 6953 Housekeeping control

21 B2M – Choroid/RPE – 6952 Housekeeping control


23 B2M – Retina – 6952 Housekeeping control


25 B2M – Sclera – 6952 Housekeeping control



28 B2M – Scleral Stromal Cells – 6952 Housekeeping control

29 B2M – Scleral Stromal Cells – 6953 Housekeeping control – Faint expression

30 Marker VIII


1 Marker VIII

2 GAPDH – Choroid/RPE – 6952 RT‐ve control

3 GAPDH – Choroid/RPE – 6953 RT‐ve control

4 GAPDH – Retina – 6952 RT‐ve control

5 GAPDH – Retina – 6953 RT‐ve control



120



9 GAPDH – Scleral Stromal Cells – 6952 RT‐ve control

10 GAPDH – Scleral Stromal Cells – 6953 RT‐ve control





25 B2M – Sclera – 6952 RT‐ve control


27 B2M – Sclera – 6954 RT‐ve control – Faint expression

28 B2M – Scleral Stromal Cells – 6952 RT‐ve control


30 Marker VIII


121

Table A18: RT‐PCR results for choroid/RPE – donor 6953 – & retina – donor 6952. The aim



Choroid/RPE (6953) & Retina (6952) – 63°C (Pages 132‐133 of Laboratory Notebook)


1 Marker VIII





6 Primer #7: GABA A Beta 1 Choroid/RPE – 6953

7 Primer #8: GABA A Beta 2v1&2 Choroid/RPE – 6953 – Weak expression

8 Primer #11: GABA A Gamma 2v1‐3 Choroid/RPE – 6953


10 Primer #13: GABA A Epsilon Choroid/RPE – 6953


12 Primer #15: GABA A Theta Choroid/RPE – 6953

13 Primer #16: GABA A Pi Choroid/RPE – 6953 – Strong expression


15 Primer #19: GABA C Rho 1 Choroid/RPE – 6953‐ Strong expression

16 Primer # 20: GABA C Rho 2 Choroid/RPE – 6953 – Strong expression









25 Primer #7: GABA A Beta 1 Retina – 6952

26 Primer #8: GABA A Beta 2v1&2 Retina – 6952 – Intermediate expression

27 Primer #11: GABA A Gamma 2v1‐3 Retina – 6952 – Strong expression



122

29 Primer #13: GABA A Epsilon Retina – 6952

30 Marker VIII


1 Marker VIII


3 Primer #15: GABA A Theta Retina – 6952

4 Primer #16: GABA A Pi Retina – 6952 – Weak expression


6 Primer #19: GABA C Rho 1 Retina – 6952 – Weak expression

7 Primer # 20: GABA C Rho 2 Retina – 6952 – Weak expression





30 Marker VIII


123

Table A19: RT‐PCR results for choroid/RPE & retina obtained from donors 6952 & 6953. The



Choroid/RPE & Retina – 6952 & 6953 – 65°C (Pages 134‐135 of Laboratory Notebook)


1 Marker VIII




5 Primer #4: GABA A Alpha 4v1‐3 Choroid/RPE – 6952 – Strong expression



8 Primer #9: GABA A Beta 3v1‐4 Choroid/RPE – 6952











19 Primer #4: GABA A Alpha 4v1‐3 Choroid/RPE – 6953 – Strong expression



22 Primer #9: GABA A Beta 3v1‐4 Choroid/RPE – 6953









124

30 Marker VIII


1 Marker VIII




5 Primer #4: GABA A Alpha 4v1‐3 Retina – 6952 – Strong expression



8 Primer #9: GABA A Beta 3v1‐4 Retina – 6952











19 Primer #4: GABA A Alpha 4v1‐3 Retina – 6953 – Strong expression



22 Primer #9: GABA A Beta 3v1‐4 Retina – 6953








30 Marker VIII


125


obtained from donor 6952. The aim of this study was to determine the presence or absence

of GABA receptors in human scleral tissue and cultured human scleral cells, as well as


Retina, Choroid/RPE, Sclera, & Scleral Stromal Cells – 6952, 3 & 4 – 63/65°C (Pages 130‐131 of Notebook)


1 Marker VIII

2 PBGD – Choroid/RPE – 6952 Housekeeping control

3 PBGD – Choroid/RPE – 6953 Housekeeping control

4 PBGD – Retina – 6952 Housekeeping control

5 PBGD – Retina – 6953 Housekeeping control

6 PBGD – Sclera – 6952 Housekeeping control – Faint expression

7 PBGD – Sclera – 6953 Housekeeping control

8 PBGD – Sclera – 6954 Housekeeping control

9 PBGD – Scleral Stromal Cells – 6952 Housekeeping control

10 PBGD – Scleral Stromal Cells – 6953 Housekeeping control










30 Marker VIII


1 Marker VIII

2 PBGD – Choroid/RPE – 6952 RT‐ve control

3 PBGD – Choroid/RPE – 6953 RT‐ve control

4 PBGD – Retina – 6952 RT‐ve control

5 PBGD – Retina – 6953 RT‐ve control

6 PBGD – Sclera – 6952 RT‐ve control


126



9 PBGD – Scleral Stromal Cells – 6952 RT‐ve control

10 PBGD – Scleral Stromal Cells – 6953 RT‐ve control










30 Marker VIII


127

Table A21: RT‐PCR results for mesenchymal stromal cells obtained from donor 6953. The



Scleral Stromal Cells – 6953 – Varied°C (Pages 143‐144 of Laboratory Notebook)


1 Marker VIII




5 Primer #4: GABA A Alpha 4v1‐3 Weak expression



8 Primer #7: GABA A Beta 1 Intermediate expression

9 Primer #8: GABA A Beta 2v1&2 Intermediate expression



12 Primer #11: GABA A Gamma 2v1‐3 Strong expression


14 Primer #13: GABA A Epsilon Intermediate expression



17 Primer #16: GABA A Pi Weak expression




21 Primer # 20: GABA C Rho 2 Weak expression


23 Marker VIII


128

Table A22: RT‐PCR results for mesenchymal stromal cells obtained from donor 6952. The



Scleral Stromal Cells – 6952 – Varied°C (Page 149 of Laboratory Notebook)


1 Marker VIII

2 Primer #1: GABA A Alpha 1v1‐7 Intermediate expression



5 Primer #4: GABA A Alpha 4v1‐3 Strong expression

6 Primer #5: GABA A Alpha 5v1&2 Strong expression


8 Primer #7: GABA A Beta 1 Strong expression

9 Primer #8: GABA A Beta 2v1&2 Strong expression


11 Primer #10: GABA A Gamma 1 Strong expression

12 Primer #11: GABA A Gamma 2v1‐3 Strong expression

13 Primer #12: GABA A Gamma 3 Weak expression

14 Primer #13: GABA A Epsilon Strong expression


16 Primer #15: GABA A Theta Strong expression

17 Primer #16: GABA A Pi Strong expression

18 Primer #17: GABA B R1 Strong expression

19 Primer #18: GABA B R2 Strong expression

20 Primer #19: GABA C Rho 1 Strong expression

21 Primer # 20: GABA C Rho 2 Strong expression


23 Marker VIII


129

Appendix Four: GABA RECEPTOR GENE SEQUENCING

Representative RT‐PCR product for each GABA receptor for which a positive result was

obtained was sent to the Australian Genome Research Facility (AGRF) for independent

sequencing. Firstly, the product was cleaned and each product was assigned an identifying

label solely (USS1‐17; no further information was supplied to the AGRF). Each label

corresponded to the different GABA receptor genes as shown below. Refer to Table A2 for

Blast results.

USS1: GABA A Alpha 1




USS5: GABA A Beta 1

USS6: GABA A Beta 2

USS7: GABA A Beta 3

USS8: GABA A Gamma 1



USS11: GABA A Epsilon

USS12: GABA A Theta

USS13: GABA A Pi

USS14: GABA B R1

USS15: GABA B R2

USS16: GABA A Rho 1

USS17: GABA A Rho 2


130

Table A23: Relevant Blast results obtained from the AGRF for each product representative of

potential positive results for GABA receptor genes.

Code USS1

Gene >gi|21265167|gb|BC030696.1| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 1, mRNA (cDNA clone MGC:26564 IMAGE:4820972), complete cds

Length 3440

Score 291 bits (147)

Expect 5e‐76

Identities 147/147 (100%)

Strand Plus/Plus

Blast Result ctggatcctccttctgagcacactgactggaagaagctatggacagccgtcattacaaga ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ctggatcctccttctgagcacactgactggaagaagctatggacagccgtcattacaaga tgaacttaaagacaataccactgtcttcaccaggattttggacagactcctagatggtta ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||tgaacttaaagacaataccactgtcttcaccaggattttggacagactcctagatggtta tgacaatcgcctgagaccaggattggg 147 ||||||||||||||||||||||||||| tgacaatcgcctgagaccaggattggg 254

Code USS2

Gene >gi|34452722|ref|NM_000809.2| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 4 (GABRA4), mRNA

Length 11143


Expect 2e‐88


Strand Plus/Plus

Blast Result aagtgtcctatgctaccgccatggactggttcatagctgtctgctttgcttttgtatttt ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||aagtgtcctatgctaccgccatggactggttcatagctgtctgctttgcttttgtatttt cggcccttatcgagtttgctgctgtcaactatttcaccaatattcaaatggaaaaagcca ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||cggcccttatcgagtttgctgctgtcaactatttcaccaatattcaaatggaaaaagcca aaaggaagacatcaaagccccctcaggaagttcccgctgctccatggcagagagag 184|||||||||||||||||||||||||||||||||||||||||||| |||||||||| Aaaggaagacatcaaagccccctcaggaagttcccgctgctccagtgcagagagag 1249

Code USS3

Gene >gi|182915|gb|L08485.1|HUMGABRA5Y Human GABA-benzodiazepine receptor alpha-5-subunit (GABRA5) mRNA, complete cds

Length 2318


Expect 6e‐63


Strand Plus/Plus

Sequence aatggagtacaccatagacgtgtttttccgacaaagctggaaagatgaaaggcttcggtt ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||aatggagtacaccatagacgtgtttttccgacaaagctggaaagatgaaaggcttcggtt taaggggcccatgcagcgcctccctctcaacaacctccttgccagcaagatctggacccc ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||taaggggcccatgcagcgcctccctctcaacaacctccttgccagcaagatctggacccc agaca 128 |||||


131

agaca 702

Code USS4

Gene >gi|109731537|gb|BC099641.3| Homo sapiens gamma-aminobutyric acid(GABA) A receptor, alpha 6, mRNA (cDNA clone MGC:116904 IMAGE:40005679), complete cds

Length 1539 Score 250 bits (126) Expect 3e-63 Identities 133/134 (99%) Strand Plus/Plus Sequence atttgatggtcagtaaa-tctggacgcctgacacctttttcagaaatggtaaaaagtcca

||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||atttgatggtcagtaaaatctggacgcctgacacctttttcagaaatggtaaaaagtcca ttgctcacaacatgacaactcctaataaactcttcagaataatgcagaatggaaccattt ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ttgctcacaacatgacaactcctaataaactcttcagaataatgcagaatggaaccattt tatacaccatgagg 135 |||||||||||||| tatacaccatgagg 473

Code USS5

Gene >gi|182918|gb|M59213.1|HUMGABRB12 Human gamma-aminobutyric acid-A (GABA-A) receptor beta-1 subunit, exon 4

Length 2165 Score 218 bits (110) Expect 4e-54 Identities 110/110 (100%) Strand Plus/Plus Sequence atacactcaccatgtatttccagcagtcttggaaagacaaaaggctttcttattctggaa

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||atacactcaccatgtatttccagcagtcttggaaagacaaaaggctttcttattctggaa tcccactgaacctcaccctagacaatagggtagctgaccaactctgggta 110 |||||||||||||||||||||||||||||||||||||||||||||||||| tcccactgaacctcaccctagacaatagggtagctgaccaactctgggta 889

Code USS6

Gene >gi|71043451|gb|BC099719.1| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, beta 2, mRNA (cDNA clone MGC:119389 IMAGE:40006779), complete cds

Length 1977 Score 127 (64) Expect 6e-27 Identities 64/64 (100%) Strand Plus/Plus Sequence acacatcaatccaggacaaaagtgacgctaaaataccttagttgctggcctatcctgtgg

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||acacatcaatccaggacaaaagtgacgctaaaataccttagttgctggcctatcctgtgg tcca 64 |||| tcca 1948

Code USS7

Gene >gi|14714964|gb|BC010641.1| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, beta 3, mRNA (cDNA clone MGC:9051 IMAGE:3871111), complete cds


132

Length 2994 Score 129 bits (65) Expect 1e-27 Identities 65/65 (100%) Strand Plus/Plus Sequence gcctcatcagtatttggactttttaaagctcgtagaaacaagacaaggtgcaccggtttc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||gcctcatcagtatttggactttttaaagctcgtagaaacaagacaaggtgcaccggtttc ataga 65 ||||| ataga 2939

Code USS8

Gene >gi|21411385|gb|BC031087.1| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, gamma 1, mRNA (cDNA clone MGC:33838 IMAGE:5289008), complete cds

Length 2264 Score 216 bits (109) Expect 5e-53 Identities 125/133 (93%) Strand Plus/Plus Sequence ggggattatgttatcatgacaannnnnnnngacctgagcagaagaatgggatatttcact

|||||||||||||||||||||| ||||||||||||||||||||||||||||||ggggattatgttatcatgacaattttttttgacctgagcagaagaatgggatatttcact attcagacctacattccatgcattctgacagttgttctttcttgggtgtctttttggatc ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||attcagacctacattccatgcattctgacagttgttctttcttgggtgtctttttggatc aataaagatgcag 293 ||||||||||||| aataaagatgcag 983

Code USS9

Gene >gi|189083761|ref|NM_198903.2| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, gamma 2 (GABRG2), transcript variant 3, mRNA

Length 4077 Score 260 bits (131) Expect 2e-66 Identities 131/131 (100%) Strand Plus/Plus Sequence ccgctgccagagtgacgctttgatggtatctgcaagcgtttttgctgatcttatctctgc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ccgctgccagagtgacgctttgatggtatctgcaagcgtttttgctgatcttatctctgc cccctgaatattaattccctaatctggtagcaatccatctccccagtgaaggacctacta ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||cccctgaatattaattccctaatctggtagcaatccatctccccagtgaaggacctacta gaggcaggtgg 131 ||||||||||| gaggcaggtgg 257

Code USS10

Gene >gi|110556626|ref|NM_033223.3| Homo sapiens gamma-aminobutyric acid (GABA) A receptor, gamma 3 (GABRG3), mRNA

Length 1677 Score 190 bits (96) Expect 8e-46


133

Identities 96/96 (100%) Strand Plus/Plus Sequence ccctgagcaccatcgccaggaagtccttgccacgcgtgtcctacgtgaccgccatggacc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ccctgagcaccatcgccaggaagtccttgccacgcgtgtcctacgtgaccgccatggacc tttttgtgaccgtgtgcttcctgtttgtcttcgccg 96 |||||||||||||||||||||||||||||||||||| tttttgtgaccgtgtgcttcctgtttgtcttcgccg 1154

Code USS11

Gene >gi|1857125|gb|U66661.1|HSU66661 Human GABA-A receptor epsilon subunit mRNA, complete cds

Length 3154 Score 274 bits (138) Expect 1e-70 Identities 144/146 (98%) Strand Plus/Plus Sequence ggtacgacgaacgcctctgttacaacgacacctttgagtctcttgttctgaatggcaatg

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ggtacgacgaacgcctctgttacaacgacacctttgagtctcttgttctgaatggcaatg tggtgagccagctatggatcccggacaccttttttaggaattctaagaggacccacgagc ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||tggtgagccagctatggatcccggacaccttttttaggaattctaagaggacccacgagc atgagatcaccatgccaacccagatg 158 |||||||||||||||| | ||||||| atgagatcaccatgcccaaccagatg 562

Code USS12

Gene >gi|5764186|gb|AF144648.1|AF144648 Homo sapiens GABA-A receptor theta (theta) mRNA, complete cds

Length 1884 Score 232 bits (117) Expect 3e-58 Identities 117/117 (100%) Strand Plus/Plus Sequence acagcaaggatgctttcgtgcatgatgtgactgtggagaatcgcgtgtttcagcttcacc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||acagcaaggatgctttcgtgcatgatgtgactgtggagaatcgcgtgtttcagcttcacc cagatggaacggtgcggtacggcatccgactcaccactacagcagcttgttccctgg485||||||||||||||||||||||||||||||||||||||||||||||||||||||||| cagatggaacggtgcggtacggcatccgactcaccactacagcagcttgttccctgg541

Code USS13

Gene >gi|2197000|gb|U95367.1|HSU95367 Human GABA-A receptor pi subunit mRNA, complete cds

Length 3282 Score 307 bits (155) Expect 9e-81 Identities 155/155 (100%) Strand Plus/Plus Sequence acaagagcttcactctggatgcccgcctcgtggagttcctctgggtgccagatacttaca

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||acaagagcttcactctggatgcccgcctcgtggagttcctctgggtgccagatacttaca ttgtggagtccaagaagtccttcctccatgaagtcactgtgggaaacaggctcatccgcc ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ttgtggagtccaagaagtccttcctccatgaagtcactgtgggaaacaggctcatccgcc


134

tcttctccaatggcacggtcctgtatgccctcaga 159 ||||||||||||||||||||||||||||||||||| tcttctccaatggcacggtcctgtatgccctcaga 615

Code USS14

Gene >gi|4063891|gb|AF099148.1|AF099148 Homo sapiens GABA-B1a receptor mRNA, complete cds

Length 3192 Score 293 bits (148) Expect 1e-76 Identities 148/148 (100%) Strand Plus/Plus Sequence gctcatccaccacgacagcaagtgtgatccaggccaagccaccaagtacctatatgagct

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||gctcatccaccacgacagcaagtgtgatccaggccaagccaccaagtacctatatgagct gctctacaacgaccctatcaagatcatccttatgcctggctgcagctctgtctccacgct ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||gctctacaacgaccctatcaagatcatccttatgcctggctgcagctctgtctccacgct ggtggctgaggctgctaggatgtggaac 148 |||||||||||||||||||||||||||| ggtggctgaggctgctaggatgtggaac 891

Code USS15

Gene >gi|4836217|gb|AF095784.1|AF095784 Homo sapiens GABA-B receptor R2 (GABBR2) mRNA, complete cds

Length 3240 Score 256 bits (129) Expect 2e-65 Identities 129/129 (100%) Strand Plus/Plus Sequence ctaccaagagctcaatgacatcctcaacctgggaaacttcactgagagcacagatggagg

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||ctaccaagagctcaatgacatcctcaacctgggaaacttcactgagagcacagatggagg aaaggccattttaaaaaatcacctcgatcaaaatccccagctacagtggaacacaacaga ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||aaaggccattttaaaaaatcacctcgatcaaaatccccagctacagtggaacacaacaga gccctctcg 129 ||||||||| gccctctcg 2749

Code USS16

Gene >gi|120659825|gb|BC130344.1| Homo sapiens gamma-aminobutyric acid (GABA) receptor, rho 1, mRNA (cDNA clone MGC:163216 IMAGE:40146375), complete cds

Length 1862 Score 194 bits (98) Expect 5e-47 Identities 101/102 (99%) Strand Plus/Plus Sequence gcatgacgtttgacggccggctggtcaagaagatctgggtccctgacatgtttttcgtgc

||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||gcatgacgtttgatggccggctggtcaagaagatctgggtccctgacatgtttttcgtgc actccaaacgctccttcatccacgacaccaccacagacaacg 102 |||||||||||||||||||||||||||||||||||||||||| actccaaacgctccttcatccacgacaccaccacagacaacg 535


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Code USS17

Gene >gi|182912|gb|M86868.1|HUMGABARHO Human gamma amino butyric acid (GABA rho2) gene mRNA, complete cds

Length 1628 Score 283 bits (143) Expect 1e-73 Identities 150/151 (99%) Gaps 1/151 (0%) Strand Plus/Plus Sequence cagcgccagca-caagagcatgaccttcgatggccggctggtgaagaagatctgggtccc

||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||cagcgccagcaacaagagcatgaccttcgatggccggctggtgaagaagatctgggtccc tgatgtcttctttgttcactccaaaagatcgttcactcatgacaccaccactgacaacat ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||tgatgtcttctttgttcactccaaaagatcgttcactcatgacaccaccactgacaacat catgctgagggtgttcccagatggacacgtg 157 ||||||||||||||||||||||||||||||| catgctgagggtgttcccagatggacacgtg 578


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Appendix Five: Genome mAPS

Shown below are representative genome maps for thirteen of the primers used for the RT‐

PCR component of this project.


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expression of gaba receptors in human scleraeprints.qut.edu.au/68604/2/emily_mcdonald_thesis.pdf ·...

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