ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1318

Structure and Function of theRetina in Children Born ExtremelyPreterm and in Children Born AtTerm

ANNA MOLNAR

ISSN 1651-6206ISBN 978-91-554-9862-7urn:nbn:se:uu:diva-317550

Dissertation presented at Uppsala University to be publicly examined in Gunnesalen,Psykiatrins hus, Ingång 10, Akademiska sjukhuset, Uppsala, Saturday, 13 May 2017 at09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination willbe conducted in English. Faculty examiner: Professor Christopher J. Lyons (Department ofOphthalmology and Visual Sciences, University of British Columbia, Vancouver).

AbstractMolnar, A. 2017. Structure and Function of the Retina in Children Born Extremely Pretermand in Children Born At Term. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1318. 79 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-9862-7.

Background: Optical coherence tomography (OCT), multifocal electroretinography (mfERG)and full-field electroretinography (ffERG) give important information about retinal structureand function.

Purpose: To collect normative data of macular Cirrus Spectral domain (SD)-OCTassessments and of mfERG measurements of healthy children (papers I and II). To assess themacular thickness with Cirrus SD-OCT and the retinal function with ffERG in 6.5-year-oldchildren born extremely preterm and in children born at term (papers III and IV).

Methods: Study participants aged 5-15 years and living in Uppsala County were randomlychosen from the Swedish Birth Register (papers I and II). In papers III and IV, the studyparticipants consisted of children born extremely preterm and children born at term – all wereaged 6.5 years. In paper III, the children were living in Stockholm and Uppsala health careregions and, in paper IV, in Uppsala health care region only. Macular thickness was assessedwith Cirrus SD-OCT and macular function with mfERG, using the Espion Multifocal systemand DTL-electrodes. The retinal function was assessed with ffERG and DTL-electrodes, usingthe Espion Ganzfield system.

Results: Altogether, 58 children participated in paper I and 49 children in paper II. In paperI, the repeatability and reproducibility of the OCT assessments were good. In paper II, theresults of the mfERG measurements were in accordance with retinal cone density and there wereno significant differences between the right and left eyes. In paper III, 134 preterm childrenand 145 children born at term constituted the study population. The central macular thicknesswas significantly thicker in the preterm group than in the control group. Within the pretermgroup, gestational age (GA), former retinopathy of prematurity (ROP) and male gender wereall important risk factors for an increased macular thickness. In paper IV, 52 preterm childrenand 45 control children constituted the study population. Significantly lower amplitudes andprolonged implicit times of the combined rod and cone responses, as well as of the isolated coneresponses, were found in the preterm group when compared with the control group. In paperIV, there was no association between GA, ROP or male gender and the ffERG assessments.

Conclusion: Normative data of Cirrus SD-OCT and mfERG assessments were reported. Theresults of the assessments were reliable. Children aged 6.5 years, born extremely preterm, had asignificantly thicker central macula and both rod and cone function were significantly reducedin comparison to children born at term. ROP had an influence on retinal structure but not retinalfunction in the present cohorts. Our results suggest that retinal development is abnormal inchildren born extremely preterm. Long-term follow-up studies are necessary in order to evaluatethe functional ophthalmological outcome in this vulnerable population of children growing uptoday.

Keywords: optical coherence tomography, multifocal electroretinogram, full-fieldelectroretinography, extremely preterm children, retinopathy of prematurity, gestational age,gender

Anna Molnar, Department of Neuroscience, Ophthalmology, Akademiska sjukhuset, UppsalaUniversity, SE-75185 Uppsala, Sweden.

© Anna Molnar 2017

ISSN 1651-6206ISBN 978-91-554-9862-7urn:nbn:se:uu:diva-317550 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-317550)

In loving memory of my grandfather Uno Börjesson

To my beloved family

Illustrations by Kari C. Toverud

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Molnar, A., Holmström, G., & Larsson, E. (2015). Macular thickness assessed with spectral domain OCT in a population-based study of children: normative data, repeatability and reproducibility and comparison with time domain OCT. Acta Ophthalmol, 93(5), 470-475.

II Molnar, A. E., Andréasson, S. O., Larsson, E. K., Åkerblom, H. M., &

Holmström, G. E. (2015). Macular function measured by binocular mfERG and compared with macular structure in healthy children. Doc Ophthalmol, 131(3), 169-176.

III Molnar, A. E., Rosén, R. M., Nilsson, M., Larsson, E. K., Holmström,

G. E., & Hellgren, K. M. (2017). Central macular thickness in 6.5-year-old children born extremely preterm is strongly associated with gestational age even when adjusted for risk factors. Retina. doi: 10.1097/IAE.0000000000001469 (Epub ahead of print).

IV Molnar, A. E., Andréasson, S. O., Larsson, E. K., Åkerblom, H. M., &

Holmström, G. E. Both rod and cone function are reduced in 6.5-year-old children born extremely preterm. Submitted.

Reprint of Paper I was made with permission from Acta Ophthalmologi-ca/Wiley. Due to publishing rules, we were unable to reprint Papers II, III and IV.

Contents

Introduction ..................................................................................................... 9

Background ................................................................................................... 11Retinal histology and function .................................................................. 11Retinal development ................................................................................. 13The fovea – the region of high visual acuity ............................................ 15Foveal development .................................................................................. 15Optical coherence tomography ................................................................. 18

Morphometry - macular thickness measurements ............................... 20Electrophysiological examinations ........................................................... 22

Full-field electroretinography .............................................................. 22Multifocal electroretinography ............................................................ 24

Preterm birth ............................................................................................. 26Retinopathy of prematurity .................................................................. 26Long-term ophthalmological outcome in children born preterm ......... 29Children born extremely preterm ......................................................... 29

Aims .............................................................................................................. 31

Materials and Methods .................................................................................. 32Statistical Methods ................................................................................... 40

Results ........................................................................................................... 42Paper I ....................................................................................................... 42Paper II ..................................................................................................... 45Paper III .................................................................................................... 48Paper IV .................................................................................................... 51

Discussion ..................................................................................................... 53Papers I and II ........................................................................................... 53Papers III and IV ....................................................................................... 55

Conclusion ..................................................................................................... 58

Sammanfattning på svenska .......................................................................... 59

Acknowledgements ....................................................................................... 64

References ..................................................................................................... 68

Abbreviations

AP-ROP Aggressive posterior ROP BW Birth weight ERG Electroretinography EXPRESS Extremely Preterm Infants Study in Sweden EPT Extremely preterm ELM External limiting membrane Fwk Foetal week FAZ Foveal avascular zone ffERG Full-field electroretinography GA Gestational age GCL Ganglion cell layer LE Left eye mfERG Multifocal electroretinography NFL Nerve fibre layer ILM Inner limiting membrane INL Inner nuclear layer IPL Inner plexiform layer IS Inner segments ISCEV International Society for Clinical Electrophysiology of Vision IQR Interquartile range OCT Optical coherence tomography ONL Outer nuclear layer OPL Outer plexiform layer OS Outer segments PL Photoreceptor layer RE Right eye RPE Retinal pigment epithelium SD Spectral domain SD Standard deviation SE Spherical equivalent TD Time domain VA Visual acuity WHO World Health Organization

9

Introduction

Early and correct diagnosis is essential in children with different ophthalmo-logical problems such as reduced visual acuity, photophobia and affected colour and night vision; it is of great importance for optimal prognosis, ha-bilitation, genetic counselling and future treatment – gene therapy, which will be available in the future (Testa et al., 2013). In childhood, there are various retinal eye diseases such as Stargardt disease, achromatopsia, X-linked juvenile retinoschisis and different types of retinal degenerations (Andréasson & Ponjavic, 1996; Eksandh et al., 2002; Eriksson et al., 2004; Fujinami et al., 2015; Kjellström et al., 2010) where both structure and func-tion of the retina are affected. In order to diagnose those eye diseases, oph-thalmological examinations with optical coherence tomography (OCT), mul-tifocal electroretinography (mfERG) and full-field electroretinography (ffERG) are of great value. These techniques provide objective information about retinal structure and function and are also valuable when monitoring the course of retinal disease. Further, examinations of children have to be painless, fast and preferably be performed without general anaesthesia. Therefore it is important to evaluate patient-friendly examination protocols suitable for children. The OCT, mfERG and ffERG devices do not provide normative data for children, so each department has to collect its own normal values from healthy children in order to compare with pathological condi-tions.

Children born preterm is another group with various types of ophthalmologi-cal problems in childhood (Holmström & Larsson, 2008; O'Connor et al., 2002). Preterm birth is also associated with structural and morphological retinal abnormalities (Maldonado et al., 2011; Rosén et al., 2015; Vajzovic et al., 2015; Yanni et al., 2012; Åkerblom et al., 2011) together with reduced electrophysiological responses in comparison to children born at term (Ecsedy et al., 2011; Fulton et al., 2005; Raffa et al., 2017; Zhou et al., 2010; Åkerblom et al., 2014). A new population of extremely preterm (EPT) chil-dren born before 27 weeks’ of gestational age (GA) is growing up as a result of advanced neonatal care and treatment (Gultom et al., 1997; Håkansson et al., 2004). The national and population-based Extremely Preterm Infants Study in Sweden (EXPRESS) has the aim to study the mortality and short- and long-term morbidity of children born before 27 weeks’ of GA (Fellman V, 2009). The increased survival of these children has been associated with

10

major neonatal morbidities, including high incidences of retinopathy of prematurity (ROP) (Austeng et al., 2009; EXPRESS Group, 2010). Follow-up studies at 2.5 and 6.5 years have revealed a greater extent of ophthalmo-logical and neurodevelopmental problems in the preterm group in compari-son to children born at term (Hellgren et al., 2016; Holmström et al., 2014; Serenius et al., 2016; Serenius et al., 2013). The retinal structure and func-tion of a population of children only consisting of extremely preterm chil-dren, however, has not been investigated to a greater extent. Such studies would hopefully contribute to a better understanding of their visual functions and lead to better prospects for habilitation.

11

Background

Retinal histology and function The retina originates from the neural ectoderm and has a complex histologi-cal organisation that is divided into ten different layers (Figure 1). The outermost layer consists of a single layer of cuboidal retinal pigment epithe-lium (RPE) cells resting on the Bruch’s membrane. The photoreceptor layer (PL) consists of the outer and inner segments of the photoreceptors. The external limiting membrane (ELM) divides the inner segment of photorecep-tors from the cell nucleus. The outer nuclear layer (ONL) consists of the cell bodies of the rods and cones. The outer plexiform layer (OPL) is a synaptic layer connecting photoreceptors to the dendrites of the bipolar cells. The following inner nuclear layer (INL) is composed of bipolar cells, horizontal cells, amacrine cells and the nuclei of Müller cells. The inner plexiform layer (IPL) is the inner synaptic layer connecting the dendrites of the bipolar cells to the ganglion cells. The ganglion cell layer (GCL) consists of the cell bod-ies of ganglion cells responsible for the transmission of signals from retina to the visual cortex in the brain. The nerve fibre layer (NFL) is made up of the axons of ganglion cells with an overlying basement membrane – the inner limiting membrane (ILM) (Björkerud, 1971; Mann, 1964). Light is converted into electrical signals by a process called phototransduc-tion. The photon is absorbed in the outer segments, the distal part of the pho-toreceptors. The outer segments contain the photon-sensitive protein rhodop-sin, which is where phototransduction occurs. Rhodopsin consists of a transmembranous protein opsin and 11-cis retinal, which is where the light absorption occurs. Photon stimulation leads to an activation of the inactive 11-cis retinal, which results in a cascade of several biochemical reactions and finally hyperpolarisation of the cell (where the membrane potential changes to a negative charge), which generates a response to the light in the form of an electrical signal. The signal is thereafter transferred to the bipolar and ganglion cell, further to the optic nerve, and finally to the brain (Baylor, 1987; Baylor et al., 1979; Yau, 1994).

12

Figure 1. Illustration of the retinal layers and cells.

Rods and cones are specialised for different types of vision, and there are important differences between them. Morphologically, the outer segments of the rods are smaller and they contain a different type of opsin. The rods are responsible for night vision (scotopic vision), are very sensitive to light (Kraft et al., 1993), and can mediate a signal from a single photon (Baylor et al., 1979). In contrast, the cones are specialised in vision under bright light (photopic vision) and are less sensitive to light (Baylor, 1987). Rods and cones mediate signals from a wide range of wavelengths. The photorecep-tors’ response to light varies in sensitivity depending on the type of visual pigment the cell contains (Kefalov et al., 2003). In the human retina, there are three types of cone that contain an opsin that makes the cell especially sensitive to long, medium and short wavelengths (Schnapf et al., 1987). A complex interaction between the signals from the photoreceptors gives the perception of colour (Jacobs, 1996).

Nerve fibre layer (NFL)

Bipolar cell

ConeRod

Horizontal cell

Ganglion cell layer (GCL)

Inner plexiform layer (IPL)

Outer plexiform layer (OPL)

Inner nuclear layer (INL)

Outer nuclear layer (ONL)External limiting membrane (ELM)

Photoreceptor layer (PL) Inner segments (IS)

Outer segments (OS)Retinal pigment epithelium (RPE)

Internal Limiting Membrane (ILM)

© K. C. Toverud

Müller cell

Amacrine cell

Ganglion cell

13

There are an average of 92 million rods and 4.6 million cones in a human eye. The distribution of rods and cones is organised into a specific mosaic pattern in the retina, which is crucial for the visual system. The rods are ab-sent in the central fovea, increase in number around the optic disc, and grad-ually decrease slowly in the periphery. The highest density of cones is found in the fovea and the number of cones decreases steeply with distance from the fovea (Curcio et al., 1990).

More than ten different morphological types of bipolar cell can be found in the mammalian retina (Euler et al., 2014; Haverkamp et al., 2003). Bipolar cells have connections with all the other neurons in the retina, and they are the link between photoreceptor signals and the ganglion cells. There are sev-eral types of cone bipolar cell, but only one type of rod bipolar cell is identi-fied, which is the most common of all bipolar cells. There are specialised pathways in the macula for cones called midget pathways, where one cone is connected to a single bipolar and a single ganglion cell (Kolb, 1995; Kolb & Marshak, 2003). Amacrine cells are interneurons expressing inhibitory neu-rotransmitters and horizontal cells interacts on the response between the photoreceptors and the bipolar cells (Balasubramanian & Gan, 2014; Jackman et al., 2011). Müller cells are glial cells that have several tasks, such as support of the metabolism for the retinal neurons and controlling the composition of extracellular space fluid (Reichenbach & Bringmann, 2013). Retinal ganglion cells are specialised neurons transferring further neuronal signals to the brain (Yu et al., 2013). The RPE has a macrophage function, supporting the neurons and transport molecules between the retina and the choroid (Steinberg, 1985).

Retinal development The retina develops over several months in both the prenatal and postnatal periods. Studies on human, monkey and mouse retinas have revealed the key stages of this development. Multipotent progenitor cells give rise to the six specialised neurons and Müller glia cells in a time-dependent order (Andressen et al., 2003). By the 6th foetal week (Fwk), the retina begins to differentiate into two primitive retinal layers: the inner neuroblastic and out-er neuroblastic layers (Mann, 1964). Around the 8th to 9th Fwk, the retinal cell differentiation starts (Mann, 1964; O'Rahilly, 1975). The inner neuro-blastic layer gives rise to ganglion cells, amacrine cells and Müller glia cells. The outer neuroblastic layer creates bipolar cells, horizontal cells and photo-receptors (rods and cones) (Mann, 1964). The creation of the ganglion cells starts first, followed by formation of cone photoreceptors, horizontal cells and amacrine cells (Figure 2) (Cepko et al., 1996; Mann, 1964; Young, 1985). Cones have been identified as early as by Fwk 8 − the same time as

14

the inner plexiform layer (IPL) is created (Hendrickson 2016). At the end of the differentiation, rod photoreceptors, Müller cells and bipolar cells are formed (Figure 2) (Cepko et al., 1996; Young, 1985). The proliferation of rods occurs in two phases; the first phase begins around Fwk 11, when the rods are identified at the edge of the fovea, and the second phase at Fwk 15, when the cell maturation occurs after rod opsin is expressed (Hendrickson et al., 2008). Hendrickson and Drucker reported that photoreceptors in the pe-ripheral retina developed earlier than in the central retina (Hendrickson & Drucker, 1992).

All retinal layers are present and most cell types can be recognised in the mid-peripheral retina at Fwk 22 (Hendrickson & Drucker, 1992). The retinal layers develop differently; a recent study by Anita Hendrickson showed that the IPL develops first and reaches the nasal and temporal peripheral edges of the eye at midgestation in contrast to the OPL, which extends much slower and reaches the periphery at Fwk 30 (Hendrickson, 2016).

The histological arrangement of retinal cells and proportions are almost adult-like at seven months after birth (Mann, 1964). The retinal morphology of a 13-year-old has been reported as being similar to that of a 37-year-old (Hendrickson & Drucker, 1992).

Figure 2: Illustration of the differentiation of progenitor cells to ganglion cells, cones, horizontal cells, amacrine cells, rods, bipolar cells and Müller cells in a time-dependent order.

Bipolarcell

Cone RodHorizontalcell

© K. C. ToverudMüller

cellAmacrine

cellGanglion

cell

Progenitor cells

15

The fovea – the region of high visual acuity The macula is the central part of the retina and measures approximately 5.5 mm in diameter. The fovea (lat: pit) centralis lies in the centre of the macula, encompasses 0.02% of the retina, and is the region of high visual acuity and colour vision. It is situated in the visual axis of the retina on the temporal side of the optic disc (Figure 3). The fovea has a special histological organi-sation in comparison to the histological features of the rest of the retina. It is characterised by a pit in the inner retina, which is centred on a area contain-ing the highest concentration of cone photoreceptors (Curcio et al., 1990) and 25% of the ganglion cells (Curcio & Allen, 1990) and the foveal avascu-lar zone (FAZ) (Gariano et al., 1994). The macula contains a macular pig-ment consisting of zeaxanthin and lutein mainly located in the photorecep-tors (Bone et al., 1988; Delori et al., 2001; Rapp et al., 2000). Previous stud-ies suggest that the pigment has an antioxidant function (Beatty et al., 2000; Rapp et al., 2000).

Figure 3. Illustration of the central retina, with the position of the macula and fovea in relation to the optic nerve (nervus (N) opticus).

Foveal development The formation of the fovea starts early in gestation and continues for a long time after birth, but the exact length of time for final maturation remains

Fovea Macula

© K. C. Toverud

N. opticus

16

unknown. Histological studies of human foetal, infant and adult eyes have elucidated the morphological process. Postnatal development has also been studied with hand-held spectral domain (SD)-OCT (Maldonado et al., 2011; Vajzovic et al., 2012). The fovea has been identified at Fwks 10-12 as a re-gion containing cone photoreceptors (Hendrickson, 2016; Hollenberg & Spira, 1972; Linberg & Fisher, 1990). At Fwks 20-22, the foveal area has been identified histologically by the presence of a single layer of cones in the ONL (Hendrickson et al., 2012; Hendrickson & Yuodelis, 1984); at this pe-riod, the ganglion cell layer, the inner plexiform layer and the inner nuclear layer are still present at the fovea. At Fwk 25, the foveal pit appears as a small depression and successively becomes wider and deeper after birth (Hendrickson et al., 2012; Hendrickson & Yuodelis, 1984; Mann, 1964; Yuodelis & Hendrickson, 1986).

There are three crucial phases that are important for the formation of fovea; first, a progressive and peripheral migration of the ganglion cell layer, the inner plexiform layer and the inner nuclear layer occurs (Figure 4). This process continues gradually after birth, creating a wider and deeper foveal pit. Secondly, the outer nuclear layer with photoreceptors becomes continu-ously thicker after a central migration of cones, resulting in tightly packed photoreceptors in multiple layers. At birth, the cones are still arranged in a single layer (Hendrickson et al., 2012; Hendrickson & Yuodelis, 1984) but, in the postnatal period the density of cones increases (Yuodelis & Hendrickson, 1986). Thirdly, the cell morphology of cones gradually chang-es and, between Fwks 25 and 38, the photoreceptors develop more distinct inner and outer segments (Hendrickson et al., 2012). After birth, the cones become more elongated and the outer segments and the basal axons (fibre of Henle) continue to develop. (Hendrickson & Yuodelis, 1984; Yuodelis & Hendrickson, 1986). At around four years of age postpartum, the fovea is morphologically adult-like, but has not the same cone density in comparison to an adult. (Hendrickson et al., 2012; Hendrickson & Yuodelis, 1984). At the age of 13-16 years, the fovea appears mature histologically and similar to that of an adult (Hendrickson et al., 2012; Vajzovic et al., 2012).

17

Figure 4. Macular development in three different stages. Stage 1: the fovea in the prenatal period - the ganglion cell layer and the inner nuclear layer are present at the fovea. Stage 2: the fovea at birth - presence of the foveal pit with a single layer of cones. Stage 3: the fovea after birth - deepening of the foveal pit and tightly packed cones.

Retinal vascularisation

In the human retina, vascularisation starts near the optic disc at 14-15 weeks of gestation. The normal development of the vasculature is stimulated by retinal hypoxia (Chan-Ling et al., 1995) and vascular endothelial growth factor (VEGF) is crucial for prenatal vasculogenesis. (Carmeliet et al., 1996). The primary vascular network (superficial vasculature) expands grad-ually in a four-lobe pattern and reaches the most peripheral extent at 37- 40 weeks of gestation (Hughes et al., 2000; Provis, 2001) (Figure 4). The sec-ondary vascular network (deep vasculature) grows from the temporal side of the optic disc and skirts around the fovea − since anti-proliferative factors prevent vessel growth, creating the foveal avascular zone (FAZ) at 25 weeks of gestation (Hughes et al., 2000; Kozulin et al., 2010; Provis & Hendrickson, 2008; Provis et al., 2000). Mintz-Hittner et al. revealed that the diameter of FAZ is correlated with GA and birth weight (Mintz-Hittner et al., 1999).

Stage 1

Stage 2

Stage 3 © K. C. Toverud

Ganglion cell layer (GCL)

Outer nuclear layer (ONL)Inner nuclear layer (INL)

18

Figure 5. Growth arrangement of the primary vasculature (a-e) in the human retina at different gestational ages (GA).

Optical coherence tomography Optical coherence tomography is non-invasive imaging using backscattering of short coherence optical radiation, and is similar to ultrasound. In 1991, OCT was first demonstrated as an imaging tool for the retina in a human eye

in vitro (Huang et al., 1991). Thereafter, Swanson et al. reported the first in vivo OCT imaging of the retina in a human volunteer (Swanson et al., 1993). Two years later, 49 patients were examined and the clinical application of the OCT device was described (Hee et al., 1995). The technique has since evolved rapidly and is now used in daily ophthalmological clinical practice. It is especially suitable for examination of children as the method is fast, non-invasive and painless (Eriksson et al., 2009; Åkerblom et al., 2011).

A retinal OCT image consists of spatially resolved intensity of backscattered optical radiation. Low-coherent, near-infrared radiation is generated from a superluminescent diode and focussed into an optical fiber, which divides the light into a sample beam and a reference beam (Huang et al., 1991). The sample beam is backscattered by the retina and the reference beam is reflect-ed by a reference mirror. The beams are then collected into the interferome-ter and allowed to interfere. If and only if the electromagnetic waves of the two beams are in close phase, superposition of the waves induces a signal that can be detected. Hence, a signal is recorded when the time-of-flight is

© K. C. Toverud

a cb

ed

14 – 15 GA

30 GA 40 GA

18 GA 22 GA

19

identical for the backscattered sample beam and for the back reflected refer-ence beam (Figure 6).

In time-domain (TD) OCT, the time-of-flight of the reflected reference beam is changed by moving the reference mirror. The interference signal is meas-ured as a function of the position of the reference mirror. The mechanical movement of the mirror limits the scanning speed. The intensity of the backscattering from various depths in the retina is referred to as an A-scan. Multiple adjacent A-scans build two-dimensional, cross-sectional images, or B-scans. Due to spatial variation of the mass density between the retinal layers, the intensity of the backscattering varies among the different layers (Chen, 1993; Chen et al., 1991). An OCT B-scan of the retina, therefore, appears as a pseudo-histological image of the retina.

In TD-OCT, six axial B-scans are collected from a central point, e.g., the fovea, to create a macular map (Figure 7). The mechanical movement of the reference mirror limits the A-scan capture speed. With Stratus TD-OCT technology (Carl Zeiss, Germany) the scan speed is 400 A-scans per second and the axial resolution is ≤ 10 µm.

Figure 6. Illustration by A.M. of the basic principle of OCT: low-coherent radiation generated from the superluminescent diode goes to the beam splitter and then to the reference mirror and the eye, back to the photodetector or spectrometer and finally on for data analysis.

20

7A 7B Figure 7. Illustration by A.M. of the OCT-scanning procedure. Figure 7A: the six radial scans around a central point −e.g., the fovea − when performing Stratus TD-OCT assessments. Figure 7B: the raster scans over the macular area when perform-ing Cirrus SD-OCT assessments. If low-coherent radiation is introduced to an interferometer, high frequencies of the sample beam travel faster than low frequencies due to dispersion of the beam. At the plane of superposition of the two beams (sample and refer-ence beams), several frequencies will interfere. The multiple interferences can be resolved with a spectrometer and further analysed mathematically with Fourier analysis. The newer technique − spectral domain (SD) OCT (Wojtkowski et al., 2002) − is 50 times faster than TD-OCT and uses a fixed reference mirror, which allows much faster data capture and also raster scans and 3D reconstructions. The axial resolution of the SD-OCT is usually 5-7 µm.

Low-coherent narrow bandwidth radiation can be frequency-tuned with a very high speed. In that way the A-scan range is increased and the capture rate becomes high. Swept-source (SS) OCT technology is the latest intro-duced technique. A fixed reference mirror is used and signal detection is performed by a photodetector. The scan speed is 249.000 A-scans per second and the axial resolution is 1 µm (Gabriele et al., 2011).

Morphometry - macular thickness measurements With all OCT devices, the quantity measured is time. Time is converted to distance assuming an average speed or refractive index in the tissue. The differences in the intensity of the backscattering are identified. In Stratus TD-OCT, a software algorithm calculates the macular thickness as the dis-tance between the vitreoretinal interface (the area between the vitreous body

21

and the retina) and the junction between the inner and outer segments of the photoreceptors. In contrast, the Cirrus SD-OCT software segmentation algo-rithm detects the ILM and RPE to calculate the macular thickness (Figure 7). Manual correction of misplaced segmentation lines is possible. Mylonas et al reported that the Stratus TD-OCT macular thickness assessments were lower in comparison to the Cirrus SD-OCT assessments (Mylonas et al., 2009), which was related to the different segmentation algorithms. Segmentation errors have been reported to be more common in eyes with retinal disease such as pigment epithelium detachment and subretinal fluid (Keane et al 2009; Song et al. 2012).

Figure 8. Marked segmentation lines of a macular SD-OCT assessment; in white on the identified ILM and in black on the detected RPE (Cirrus version 6.0.2.81, Carl Zeiss Meditec Inc., Dublin, CA).

Previous studies have shown that there is a good agreement between macular OCT images and retinal morphology (Toth et al., 1997; Vajzovic et al., 2012). Measurements of the total retinal thickness in human tissues in com-parison to OCT assessments have also been reported as being in agreement (Chauhan DS, 1999). A nomenclature system for the classification of the retinal layers assessed with SD-OCT has been suggested by retinal special-ists (Staurenghi et al., 2014). The retinal layers were described as hyper- or hypo-reflective layers or bands on the OCT image and were thereafter identi-fied as corresponding retinal structures.

22

Electrophysiological examinations Electrophysiological examinations give important, objective information of the total and local electrical retinal responses. Electroretinography (ERG) is a valuable investigation method for a wide range of patients with different ophthalmological problems, where retinal and/-or macular diseases are sus-pected. Further, hereditary retinal diseases can be diagnosed and monitored with this method. The examination can be performed on children without general anaesthesia from about 5-6 years of age.

Full-field electroretinography Full-field electroretinography (ffERG) is an electrophysiological test meas-uring the total electrical response to light in the retina. It originated from a finding by Fritihof Holmgren in 1865 when he discovered that there was a change in the electric potential of a frog’s eye when it was stimulated with light (Holmgren, 1865). Mass field potentials are generated from a summa-tion of the extracellular ion composition around the neurons and glia cells in the retina. These electrical signals are propagated to the cornea and can be measured with electrodes. Since the mid-80s, the ffERG has been used in clinical practice. The Interna-tional Society for Clinical Electrophysiology of Vision (ISCEV) has created six standardised ERG protocols for ffERG, so that examinations can be per-formed in similar settings and become comparable around the world (McCulloch et al., 2015). A Ganzfeld (full-field) bowl is placed around the front of the head and eyes of the patient to stimulate the whole retina with equable luminance (Figure 9). Different ERG protocols are based on the variation of the flash strength, frequency, duration and wavelength, together with the state of previous adaptation of the eye, i.e., dark or light adaptation. Flash strengths are expressed in candela-seconds per square metre (cd.s/m2), i.e., luminous energy per steradian and per square metre. The rod and cone response can, in that order become isolated and analysed separately. Previ-ous dark adaptation and stimulation with a low flash strength give rise to a rod response. When the flash strength successively increases, a combined rod and cone response is obtained. Finally, the isolated cone response is rec-orded after light adaptation and stimulus with high flash strength. The stimu-lation of the eye can either be performed monocularly or binocularly.

23

Figure 9. Picture of the Ganzfeld dome and the chin rest setting just before making the ffERG recordings. The bowl is moved closer to the patient when the measure-ments start (published with permission from study participant and parent).

The electrodes can be placed on either the cornea, conjunctiva, or skin on the lower eyelid. Depending on which electrode is in use, the amplitude varies because of the different impedance in the electrodes, but the ERG wave forms are similar (Bradshaw et al., 2004). Reference electrodes can be placed on the skin over the cheekbones (zygomatic bones) and the ground electrode on the back of one hand. The skin is lightly abrased and prepared with conductive gel before the electrode placement since low impedance is crucial for obtaining reliable signals. In this thesis, the a-wave and the b-wave of the ffERG response were analysed. The a-wave, which is cornea-negative, is considered to measure the response from the photoreceptor (Brown & Watanabe, 1962; Brown & Wiesel, 1961). The cornea-positive b-wave originates primarily from the bipolar cells (Robson & Frishman, 1998; Stockton & Slaughter, 1989) (Figure 10). The ERG signals are embedded in a high amount of noise and therefore amplifiers and band-pass filters are necessary for increasing the level of the signal and for identifying the correct sine waveform signals. An artefact signal is a signal with an abnormal ampli-tude caused by movements or blinks; it can be filtered away with an artefact rejection system.

24

Figure 10. Illustration of the dark-adapted ffERG waveform, the amplitude (µv) and implicit time (ms) of the a- and b-waves.

Multifocal electroretinography Multifocal electroretinography (mfERG) provides assessments of the local retinal function. The technique was developed by Erich E. Sutter and Duong Tran in 1990 (Sutter & Tran, 1992). The eye is stimulated under light-adapted conditions by a flickering hexagonal stimulus on a screen with a central fixation cross. The hexagons in the centre are smaller than in the periphery. Good fixation during the test is absolutely essential for obtaining correct mapping of the macula. The hexagons flicker in a determined math-ematical sequence called the pseudorandom binary m-sequence, which is correlated with the obtained bioelectrical potentials, and several local ERGs can be created. Hence, a topographic measure of the macula and central reti-na is provided. The number of hexagonal elements can vary between 37 to 241, depending on the extent of retinal mapping that is required. The more hexagons, the larger retinal area, (up to 25° radius from the central fixation point) that can be mapped. The luminance of the stimulus is expressed in candela-seconds per square meter (cd.s/m2), i.e., luminous energy per stera-dian and per square metre. The stimulation of the eye can be either monocu-lar or binocular and as with ffERG, different electrodes can be used. Refer-ence electrodes and a ground electrode are used. As with ffERG, low elec-trode impedance is crucial for obtaining reliable signals and a high signal-to-noise ratio. The mERG signals are amplified and band-pass filtered in order to detect reliable signals as with ffERG. Spatial averaging is another kind of filter that mfERG devices use in order to reduce noise and create smooth waveforms, which means an averaging of the mfERG waveform with a per-centage of the waveform from the neighbouring signal. Like the ffERG, the software uses artefact rejection in order to avoid distorted signals. A stand-ardised protocol for basic mfERG has been created from the ISCEV (Hood

25

et al., 2012). The mfERG waveform consists of two troughs (N1, N2) and one positive peak (P1) (Figure 11) and is referred to as the first order kernel. In this thesis, the P1 was analysed. The presented responses are not the direct bioelectrical potentials but mathematical extractions from signal. The origin of the signal is predominantly cone-driven and primarily from bipolar cells (Hood et al., 2002; Hood et al., 1999).

Figure 11. The mfERG waveform with the implicit time (ms) and amplitude (nV) of the first positive peak (P1) and the two throughs (N1) och (N2).

26

Preterm birth

According to the World Health Organization (WHO), prematurity is defined as birth before 37 weeks’ gestation and extreme prematurity as birth before 28 weeks’ gestation. The time period between the first day of the last men-strual period and the day of delivery is called GA. Ultrasound has been re-ported as a reliable method for determining the length of the pregnancy (Tunon et al., 1996). In Sweden, ultrasound is routinely used for determining the GA.

Preterm birth is multifactorial. In the case of spontaneous preterm labour, various mechanisms can be involved, for example infection or inflammation. The delivery can be induced or initiated by Caesarean section in the case of maternal conditions such as infection or preeclampsia. Multiple pregnancies, as well as in-vitro fertilisation, are also associated with a higher risk of prematurity (Jackson et al., 2004; Klebanoff & Keim, 2011). Further, previ-ous preterm delivery has been reported as entailing an increased risk of pre-term birth in the next pregnancy (Mercer et al., 1999). Advanced neonatal care, treatment with corticosteroids during the pregnan-cy, assisted ventilation and surfactant therapy have all contributed to a major increase in the survival of preterm children since 1990, especially those born between 23-27 weeks of GA (Gultom et al., 1997; Håkansson et al., 2004). The increased survival has been associated with a high extent of neonatal morbidities (de Kleine et al., 2007). In the neonatal period the premature infant may develop various morbidities such as chronic lungdisease (bron-chopulmonary dysplasia), intracranial haemorrhage (intraventricular haem-orrhage), ischemia in the white matter (periventricular leukomalacia), in-flammatory bowel disease (necrotising enterocolitis) and retinopathy of prematurity (ROP), (Lagercrantz et al., 2015).

Retinopathy of prematurity The first incubator, which could maintain high concentrations of oxygen for newborn infants with neonatal hypoxia, became available at the beginning of the 1940s (James & Lanman, 1976). Thereafter, the first cases of ROP were reported and named retrolental fibroplasia (Terry, 1942). The relationship between oxygen supply in incubators and ROP was first described in 1952 ((Patz et al., 1952). Oxygen therapy increased the survival of the preterm infants, but was at the same time a risk factor for developing ROP (Walsh et al., 2009). Several studies over the following decades have shown that ROP is a multifactorial disease, and that two other major risk factors are low GA and low birth weight (BW) (Holmström et al., 1993). Further, low insulin-

27

like growth factor 1 (IGF-1) levels after birth have also been reported as being associated with the disease (Hellström et al., 2003). The IGF-1 levels increase slowly in the preterm infant in comparison to infants born at term (Lineham et al., 1986) Other identified risk factors for ROP are neonatal bacteraemia (Tolsma et al., 2011) and hyperglycaemia (Garg et al., 2003). There also seems to be a relationship with ethnicity where black maternal origin is associated with a reduced risk of developing severe ROP (Husain et al., 2013). ROP is a main cause of blindness in childhood worldwide. In the middle-income countries of Latin America, for example, blindness is caused by ROP in up to 60% (Gilbert, 2007). In Sweden, around two children per year be-come blind due to ROP (according to the Swedish national register for ROP, SWEDROP, personal communication G Holmström).

Pathogenesis The exact pathophysiologic mechanism of ROP is still unknown, although abnormal retinal vessel development has been identified as the main cause of the disease. Two phases of ROP have been described (Chan-Ling et al., 1992); in the first phase, there is an inhibition of vascularisation as a result of hyperoxia and inhibition of the vascular endothelium growth factor (VEGF) (Alon et al., 1995). The growth of vessels stops and a demarcation line in the retina appears initially. The preterm infant is born with a retinal peripheral avascular zone since the retinal vessels have not yet extended to the retinal periphery (Provis, 2001; Provis et al., 1997). Progressive maturation of the retina causes hypoxia, due to the high metabolic activity in the tissue, which stimulates further vascularisation (Chan-Ling et al., 1995). In the second phase, there are increasing levels of VEGF resulting in neovascularisation, which subsequently can lead to retinal detachment (Aiello et al., 1995; Provis et al., 1997; Smith et al., 1994).

Classification, treatment and screening ROP is divided into five stages, according to the International Committee for the Classification of Retinopathy of Prematurity (International Committee for the Classification of Retinopathy of Prematurity, 2005). For description of the localisation of ROP, the retina is divided into three zones: zone I is located in the central retina and has a radius of double the distance from the optic disc to the macula, zone II extends to the nasal ora serrata, and zone III to the temporal ora serrata. ROP stage 1 is characterised by a demarcation line between the vascular and avascular part of the retina, which progresses to a ridge in ROP stage 2. In stage 3, there are fibrous and vascular proliferations and in stage 4 there is a partial retinal detachment. ROP stage 5 is characterised by a total retinal

28

detachment (Figure 12). ‘Plus disease’ is a condition with dilated and tortu-ous vessels and is a severe sign of progressive disease. Finally, aggressive posterior ROP (AP-ROP) is a serious type of ROP, which in most cases pro-gresses to ROP stages 4 and 5, if not treated in time.

Figure 12. The five different stages of ROP. Stage 1: demarcation line between the vascular and avascular part of the retina. Stage 2: ridge. Stage 3: extraretinal fi-brovascular proliferations. Stage 4: partial retinal detachment. Stage 5: total retinal detachment. Treatment for ROP is performed according to the recommendations of the Early Treatment for ROP study (Early Treatment For Retinopathy of Prematurity Cooperative, 2003) Laser photocoagulation is the treatment of choice today, replacing the earlier treatment of cryotherapy. In recent years, intravitreal treatment with anti-VEGF injections has been used, mainly for ROP in zone 1 and posterior zone II (Mintz-Hittner et al., 2011); this treat-ment is still considered controversial due to limited knowledge about the correct dosage, long-term follow-up and any the ocular and systemic side effects.

Screening for ROP in the neonatal period is crucial for detecting ROP re-quiring treatment and in order to perform treatment in time. Population-based studies have resulted in the creation of guidelines for the screening procedure of ROP in Sweden (Holmström et al., 1993; Larsson & Holmström, 2002). The Swedish national register for ROP (SWEDROP), established in 2006, has the aim of improving and evaluating routines for the screening and treatment of ROP. In Sweden, screening of ROP starts at ≤ 30

Stage 1

Stage 4 Stage 5

Stage 2 Stage 3

© K. C. Toverud

29

weeks of GA (upper limit: 30+6 days) or at a birth weight ≤1500 grams if the GA is uncertain. Follow-up studies have shown that the screening proce-dure is safe and incidences of ROP, as well as treatment frequencies are sim-ilar during a study period of five years. (Holmström et al., 2015, 2016).

Long-term ophthalmological outcome in children born preterm Population-based, long-term ophthalmological follow-up studies have re-vealed that children born preterm have a higher risk of ophthalmological sequelae than children born at term. However, there is still no national or international consensus on the follow-up procedure or screening of these children.

The visual system in the preterm child can be affected in many ways. Chil-dren born preterm have lower visual acuities at distance and near, reduced contrast sensitivity, higher risk for visual field loss, and a higher prevalence of strabismus and refractive errors in comparison to children born at term (Fledelius, 1996; Holmström et al., 1999; Holmström & Larsson, 2008; Larsson et al., 2003; O'Connor et al., 2002). Visual dysfunction in the pre-term child is mainly caused by ROP and neurological disease. ROP has also been associated with decreased central visual function and predominantly a reduced rod function (Bowl, Lorenz, et al., 2016; Fulton et al., 2005; Fulton et al., 2001; Hamilton et al., 2008). McLoone et al. reported significantly lower distance visual acuity in children who received laser treatment for ROP in comparison to control children in a long-term follow-up study (McLoone et al., 2006). Structural abnormalities have also been associated with ROP such as shallow anterior chamber (Wu et al., 2012), reduced reti-nal nerve fibre layer thickness (Åkerblom et al., 2012), a thick central macu-la (Wu et al., 2012) and presence of preretinal membranes (Chavala et al., 2009). Myopia is also more frequent in children with previous ROP (Quinn et al., 2013). Neurological disease, such as periventricular leukomalacia can lead to cerebral visual impairment and nystagmus in the preterm child (Jacobson et al., 1998; Jacobson & Dutton, 2000). Further, IVH and cerebral palsy has been associated with poor visual function (Ospina et al., 2005). It should also be mentioned that, although ROP and neurological disease are the main causes of visual impairment, prematurity per se is a risk factor for visual dysfunction (Holmström & Larsson, 2008; Raffa et al., 2017).

Children born extremely preterm Several large population-based studies of children born extremely preterm have been performed (Azria et al., 2016; Kamper et al., 2004; Markestad et al., 2005; Sutton & Bajuk, 1999; Vanhaesebrouck et al., 2004; Wood et al., 2000) but very few with a focus on the long-term ophthalmological outcome

30

(Fledelius et al., 2015; Haugen et al., 2012). According to the Swedish medi-cal birth register, over 400 children are born extremely preterm every year (0.4% of all births). The national and population-based Extremely Preterm Infants in Sweden Study (EXPRESS) (EXPRESS Group, 2010; Fellman V, 2009) studies the mortality, morbidity and the short- and long-term outcomes of extreme prematurity. All children born extremely preterm in Sweden be-fore 27 weeks of GA (n=1011) between April 1, 2004 to March 31, 2007 were included in the study. The overall one-year survival was 70% for the children born alive (Fellman V, 2009). All surviving infants needed inten-sive care and, at birth, 60% were intubated and 85% needed mechanical ven-tilation during the first period of their hospitalisation (EXPRESS Group, 2010). Intubation at birth was more frequent in infants with a GA of 23-25 weeks than in infants born at 26 weeks of GA (Fellman V, 2009). In the EXPRESS study cohort, ROP was diagnosed in 73% and 20% were treated for it (Austeng et al., 2009; Austeng et al., 2010). Overall, major neonatal morbidities were found in many of the infants: 34% had ROP ≥ stage 3, 25% had severe bronchopulmonary dysplasia, 10% had intraventricular haemor-rhage ≥ grade 3, 5.8% had necrotising enterocolitis, 5.7% had periventricular leukomalacia (EXPRESS Group, 2010). At the 2.5-year follow-up, one-third of the children born extremely preterm had ophthalmological problems such as visual impairment, major refractive errors or strabismus (Holmström et al., 2014). Further, the follow-up showed that children born extremely pre-term had lower cognitive, communicative and motor function skills than children born at term. (Mansson & Stjernqvist, 2014; Serenius et al., 2013). The neurodevelopmental outcome improved with increasing gestational age. At the 6.5-year follow-up, 38% of the children born extremely preterm had major ophthalmological problems (Hellgren et al., 2016) and 30% had mod-erate or severe cognitive disability. Overall, the presence of disabilities were more common in children born extremely preterm in comparison to children born at term (Serenius et al., 2013).

31

Aims

• To collect normative data of macular Cirrus SD-OCT assessments of healthy children aged 5-15 years, born at term, and to compare the results with macular Stratus TD-OCT measurements. To investigate the repeatability and reproducibility of Cirrus SD-OCT assessments (Paper I).

• To collect normative data of binocular mfERG measurements of healthy children, aged 5-15 years and born at term. To compare the-se measurements with macular Cirrus SD-OCT assessments (Paper II).

• To assess the macular thickness with Cirrus SD-OCT of 6.5-year-old children born extremely preterm and control children born at term who are participating in the EXPRESS study. To investigate risk factors associated with the macular thickness in the preterm group (Paper III).

• To assess the retinal function with binocular ffERG of 6.5-year-old children born extremely preterm, compared with control children born at term who are participating in the EXPRESS study. To inves-tigate the impact of GA and ROP on the retinal function in the pre-term group (Paper IV).

32

Materials and Methods

Papers I and II Study participants (n=200) aged 5 to 15 years and living in Uppsala County were randomly chosen from the birth register of the Swedish National Board of Health and Welfare. The inclusion criteria from the birth register were being born ≥ 37 weeks of gestation and a birth weight ≥ 2500 grams (g). A recruitment letter was sent out to the children and their parents. Written con-sent from the parents and oral consent from the children were required to participate. The children were specifically asked whether they wanted to participate in examinations with OCT and mfERG respectively. Ethical ap-provals for the studies were obtained from the Regional Ethical Board of Uppsala in Sweden, and the studies were performed in accordance with the Declaration of Helsinki. A medical and ophthalmic history was initially tak-en for each participant. Monocular visual acuity testing of the right and left eyes (REs and LEs) was performed with LogMAR charts. If the study partic-ipant couldn’t read letters, an HVOT chart was used (Hedin & Olsson, 1984). The pupils were dilated with phenylephrine 1.5% and cyklopentolate 0.85% eye drops. Fundus examinations with slit lamp and autorefraction in cycloplegia were performed. The inclusion criteria for participating in the studies were: being in good health, normal fundus, visual acuity of ≤ 0.1 LogMAR (≥ 0.8 decimal acuity), and refraction with a spherical equivalent of between +3.0 and -3.0, and an astigmatism < 2.0 dioptres.

Paper I The macular thickness was assessed with SD-OCT Cirrus, version 6.0.2.81 (Carl Zeiss Meditec Inc., Dublin, CA), and thereafter with TD-OCT Stratus OCT 3, version 4.0.1 (Carl Zeiss Meditec Inc., Dublin, CA). The macular cube 512x128 (512 horizontal B-scans and 128 vertical B-cans) protocol was used with Cirrus SD-OCT, and the fast macular map protocol was used with the Stratus TD-OCT. For Cirrus SD-OCT, three examinations of the macula were performed by A. Molnar (A.M.) and thereafter by a research nurse E. Nuija (E.N.). Three measurements with the Stratus TD-OCT were then ob-tained by E.N. The inclusion criteria for the OCT scans were: scanning from the central point of the macula − i.e., the fovea, a signal strength of > 7, no

33

artefacts, no great vertical or horizontal eye movements and no blinks dis-torting the signal over the measured area. The signal strength was a meas-urement of the amount of backscattered radiation, where 10 was the highest amount and ≥ 6 was generally required for a good OCT image. The macular thicknesses (µm) of the nine ETDRS areas (Early Treatment Diabetic Retinopathy Study Research Group, 1985) was used (Figure 13). The areas represented different locations in the macula − A1: the central area, A2: in-ner superior area, A3: inner temporal area, A4: inner inferior area, A5: inner nasal area, A6: outer superior area, A7: outer temporal area, A8: outer infe-rior area, A9: outer nasal area. The inner circle area was defined as the re-gion consisting of A2 to A5 and the outer circle area as the area A6 to A9. The central area A1 had a diameter of 1 mm. The total diameter of A1 and the inner circle was 3 mm. The total diameter of A1 and the inner and outer circle was 6 mm. The macular average thickness in µm (CAT: cube average thickness) and the total macular volume in mm2 (CV: cube volume) were also assessed. The study participants had to have OCT assessments accord-ing to the inclusion criteria in at least one eye to be included in the data analyses. EDTRS areas not fulfilling the inclusion criteria in one OCT exam-ination − for example, being distorted by a movement or a blink − were not included in the study. Each OCT scan was inspected after the examinations by A.M., with an evaluation of the segmentation procedure ensuring it was correct and that the inclusion criteria were followed for each OCT scan and ETDRS area before the statistical analyses.

Figure 13. Templates of the nine ETDRS areas of the OCT assessments in the right eye and the left eye. A1: the central area, A2: inner superior area, A3: inner tempo-ral area, A4: inner inferior area, A5: inner nasal area, A6: outer superior area, A7: outer temporal area, A8: outer inferior area, A9: outer nasal area.

34

Paper II The mfERG device Espion Standalone Multifocal System (Diagnosys, Low-ell, MA, USA version 6.2012.1211.52) was used for the examinations. The assessment was adapted to a paediatric population using DTL-electrodes (Dawson et al., 1979) which were made of silver-nylon threads and were placed over the lower eyelids with previous installation of anaesthetic eye drops (Tetracain 1%). Reference electrodes were placed on the skin over the zygomatic bones and a ground electrode on the back of one hand (Figure 14). The skin was carefully abrased before the electrode placement. A hex-agonal stimulus of 37 hexagonal elements corresponding to 20 degrees from the centre of the macula, i.e. the fovea, with a central fixation cross was used (Figure 15 and Figure 16).

Figure 14. Picture of the DTL and reference electrodes on one of the study partici-pants. The electrodes were placed over the lower eyelids and the reference elec-trodes over the cheekbones. (Publication of this picture was agreed by the study participant and parent).

Figure 15. Illustration of the mfERG stimulus with 37 hexagonal elements and the central red fixation cross.

35

Figure 16. Illustration of the 37 hexagonal elements in relation to the measured retinal area in degrees from the central point of the macula, i.e., the fovea. Picture approved for reuse by Springer Nature from the published article by Molnar et al. in Doc Ophthalmol.(2015;131[3]:169-76).

The sizes of the hexagons were scaled for cone density in the macula, with larger hexagons in the periphery. A liquid crystal display (LCD) presented the stimulus with a luminance of 200 cd.s/m2 and a stimulation rate of 75 Hz for the hexagonal elements. The examinations were performed according to ISCEV recommendations (Hood et al., 2012) except for the hexagonal stim-ulus with 37 hexagons. The reason for using fewer hexagonal elements was to adjust the method to a paediatric population. The viewing distance was fixed at 330 mm. MfERG was recorded binocularly using DTL-electrodes. Before the recordings, the pupils were checked to ensure they were fully dilated and that the child was sitting in a relaxed position with their eyes at the correct height in relation to the red fixation cross. The impedance of the electrodes was checked and a value of less than 10 kΩ was accepted before making the recordings. The examination time was around 2 minutes if the test was performed without pause and a high number of blinks. The fixation was checked with a camera focusing on the eyes during the recordings, and the examiner encouraged the child to fixate on the red fixation cross throughout the whole test. The majority of the mfERG examinations were performed by A.M.. The signals were filtered through a 10-100 Hz band-pass filter and spatial averaging of 25% was used. The recordings were am-plified with 32-bit amplifiers. The amplitudes and implicit times of the first positive peak, P1, were recorded. The hexagonal elements were divided into three areas: Ring 1, Ring 2 and Ring 3 (Figure 17).

36

Figure 17. Illustration of the mfERG Rings 1-3 without scaling for cone density. Ring 1: orange, Ring 2: green, Ring 3: pink. Picture approved for reuse by Springer Nature from the published article by Molnar et al., Doc Ophthalmol. (2015;131[3]:169-76).

Rings 1 and 2 measured the macular area and Ring 3 the retina outside the macula. The response density of P1 was also recorded; the amplitudes in a Ring were divided by the area of the hexagons in that Ring using the formula nanovolts per square degree (nV/deg2). The mfERG measurements were presented in trace arrays with the amplitudes and 3D topography for illustrat-ing the response density for the RE and the LE (Figure 18).

Figure 18. The mfERG measurements in one of the study participants. Trace arrays showing the amplitude and 3D topography of the response density of the RE and LE. Picture approved for reuse by Springer Nature from the published article by Molnar et al., Doc Ophthalmol. (2015;131[3]:169-76).

37

The OCT assessments were performed three times in a row by A.M. or E.N. with the SD-OCT Cirrus, version 6.0.2.81 (Carl Zeiss Meditec Inc, Dublin, CA. The OCT assessments were divided into the nine ETDRS areas, exactly as in Paper 1. The inclusion criteria for the OCT scans were identical as for paper I. Each mfERG measurement was inspected by A.M. after the exami-nation, excluding mfERG recordings with distorted signals due to alternating current disturbances or poor cooperation from the study participant. Each OCT scan was reviewed as in paper I.

Papers III and IV The study population was comprised of 6.5-year-old children born extremely preterm (born before 27 weeks of GA) and control children; all children were born between April 1, 2004 and March 31, 2007 and were participating in the EXPRESS study (EXPRESS Group, 2010; Fellman V, 2009). The children featured in paper III were from Stockholm and Uppsala health care regions. In paper IV, the children were from Uppsala health care region. The control children were born at term (GA ≥ 37 weeks) with normal birth weight (≥ 2500 grams (g)) and were both geographically and age-matched with the preterm group. The regional ethics boards of Lund University, Stockholm University approved papers III and IV, which were carried out in accordance with the Declaration of Helsinki. Written consent from the par-ents and oral consent from the children were required for the participation. The children born extremely preterm (EPT) underwent screening for ROP neonatally (Austeng et al., 2009). The guidelines of the International Com-mittee for the Classification of Retinopathy of Prematurity Grading of ROP were followed: stage 1 and stage 2 were defined as mild ROP and stages 3 to 5 as severe ROP (International Committee for the Classification of Retinopa-thy of Prematurity, 2005). The recommendations of the Early Treatment for ROP study were used as a guideline for the treatment (Early Treatment For Retinopathy of Prematurity Cooperative, 2003) (Austeng et al., 2010). Re-garding presence of intraventricular haemorrhage (IVH), classification and grading were performed according to Paplie et al (Papile et al., 1978): mild IVH was defined as stages 1 and 2 and severe IVH as stages 3 and 4. Mo-nocular visual acuity (VA) testing in REs and LEs was performed with ha-bitual correction using LogMAR Lea Hyvärinen (LH) symbols charts (Hyvärinen et al., 1980). The spherical equivalent (SE) was calculated from refraction measurements in an automated refractor in cycloplegia (using dilating eye drops as in papers I and II). The examinations were performed between 2010-2014.

38

Paper III The macular thickness was assessed at least once until obtaining a measure-ment according to the inclusion criteria in both eyes with SD-OCT Cirrus – software versions 5.1.1.6, 5.1.0.96, 6.0.2.81 and 6.5.0.772 (Carl Zeiss Medi-tec Inc., Dublin, CA, USA) − using the same macular cube protocol as in papers I and II. The assessments were divided into the nine ETDRS areas as in papers I and II. The inclusion criteria for an OCT assessment were stated in papers I and II, where a signal strength of ≥ 6 was accepted. The study participants had to have OCT measurements fulfilling the inclusion criteria in at least one of their eyes to be included in the analyses. Each scan was inspected after the examinations in Uppsala by A.M. and in Stockholm by R. Rosén (R.R.) and M. Nilsson (M.N.), ensuring that the segmentation was correct and that the inclusion criteria were followed for each scan.

Paper IV FfERG was performed with the Espion Ganzfield system (Diagnosys, LCC Lowell, MA, USA) according to the ISCEV standards (McCulloch et al., 2015), except the light-adaption time, which was excluded to adapt the ex-amination for a paediatric population. FfERG assessments were performed binocularly with DTL electrodes. The procedure for placement of DTL and reference electrodes was exactly the same as in paper II. An impedance of less than 10 kΩ was required before the recordings were done. Pupils were fully dilated with eye drops as in paper II. Six ffERG protocols with differ-ent flash strengths from LEDs light sources were used (Table 1). The unit candela per square meter (cd.s/m2) defined the light intensity of the flashes. Nomenclatures for each protocol were stated (Table 1). Dark adaptation was performed for 20 minutes.

39

Table 1. The six ffERG protocols with different flash strengths in luminance (cd.s/m2), ffERG nomenclature and whether dark adaptation was performed before the recordings is shown, together with the type of photoreceptor response for each protocol. Flash strengths (cd.s/m2)

Nomenclature Type of photoreceptor response

0.009 0.17 3.0 12.0 3.0 3.0

Dark-adapted 0.009 ERG Dark-adapted 0.17 ERG Dark-adapted 3 ERG Dark-adapted 12 ERG 30 Hz flicker ERG 3 single cone flash ERG

Rods Rods/Cones Rods/Cones Rods/Cones Cones Cones

Cd.s/m2= candela-seconds per square metre Each ERG protocol was assessed with six measurements in a row. The soft-ware did an automatic mean calculation of all six recordings, creating the final ERG curve from which the implicit time and amplitude were measured. The protocols 30 Hz flicker and 3.0 single cone flash ERG were performed with a background dome light of 34 cd.s/m2 in room light. The a-wave was measured and analysed in the combined rod and cone protocols: 0.17, 3.0 and 12.0 ERG. An amplitude greater than 1 µV was required for inclusion. The b-wave was assessed and analysed in all ERG protocols. The b-wave of the 30 Hz flicker was defined as the peak of the wave. The ffERG measure-ments were filtered through a band-pass filter of 0.3-300 Hz and artefact rejection excluding responses >1000 uV was used. Each study participant had to be seated in a relaxed position before the assessments started. Eye fixation and detection of larger eye movements and/or blinks were moni-tored with an infrared camera in the Ganzfeld dome and evaluated continu-ously during the test by the examiner. Each child was encouraged to follow procedure throughout the ERG recordings. The inclusion criteria for the measurements were: good cooperation without large eye or body move-ments, or blinks, and no alternating current disturbances distorting the signal. Each ERG protocol was reviewed by H. Åkerblom (H.Å.) and A.M. ensur-ing the inclusion criteria were followed before the statistical analyses.

40

Statistical Methods All statistical analyses were performed after discussions with, or by a profes-sional statistician. Model validations were performed for all statistical tests and considered acceptable before starting the analyses. For the statistical analyses, the SPSS (Statistical Package for Social Sciences, IBM Corp, Ar-monk, NY, USA) program was used (version 21 in papers I and II and ver-sion 22 in papers III and IV) and also the R program (version 3.1.1 in papers II and III and version 3.2.3 in paper IV). For all analyses, a p-value of <0.05 was chosen as the significance level. In all papers, no adjustment for multi-plicity was performed so the p-values should be interpreted as descriptive.

Paper I The Kolmogorov-Smirnov test was done in order to verify whether the data was normally distributed. The mean values and standard deviations were calculated for the three OCT assessments performed by A.M. and E.N. A paired sample T-test was used to compare the REs and LEs and also when comparing the SD-Cirrus and TD-Stratus OCT assessments. A randomisa-tion of one eye from each child was performed in order to present the macu-lar thickness (EDTRS areas 1-9, CAT, CV the inner- and outer circle). Pear-son’s product moment correlation was used when performing correlation between the eyes and between the randomised eye and VA, SE and age. An independent sample T-test was used to compare the macular thickness in girls and boys in the randomised eye. Regarding the inner macular circle, a multiple linear regression analysis, including age and gender was used for the randomised eye. Repeatability (intra-observer variation) and reproduci-bility (inter-observer variation) were analysed as a coefficient of variance (CoV) and an intraclass correlation (ICC) in the randomised eye.

Paper II The recommendations from ISCEV (Hood et al., 2012) were followed and non-parametric tests were used. The median values and interquartile ranges (IQR) for the mfERG measurements (implicit time, amplitude and response density) were calculated. The Wilcoxon signed-rank test was used for com-parison of the REs and LEs. Friedman rank sum test was used to compare the mfERG measurements for each mfERG ring (1-3). The Mann-Whitney U-test was used to compare girls and boys for each mfERG value. The Spearman rank order correlation test was used for correlation of the mfERG values and age as well as the macular thickness (EDTRS areas 1-9, CAT, CV the inner- and outer circle). An analysis of covariance (ANCOVA) was done to explore the effects of age and gender on implicit time.

41

Paper III The mean values and standard deviations were calculated for the macular thickness (EDTRS areas 1-9, the inner- and outer circle). An independent sample T-test was used when comparing the mean values of continuous data and presented with 95 % confidence intervals. Several series of linear mixed models were used for further analyses of the macular thickness. The preterm group was compared with the control group separately for A1 and the inner and outer circle using the eye (RE and LE) and group (preterm and control group) as fixed factors and the subjects as random factors. The main effects of the group differences were presented with corresponding 95 % confidence intervals. The preterm group was then analysed separately. In order to evalu-ate factors related to A1 and the inner and outer circles, analyses were per-formed in two steps. In the first step, simple (univariable) linear mixed mod-el analyses were performed for the independent factors (GA, BW, gender, presence of previous ROP [present or not, irrespective of stage or treatment]) and no, mild (stages 1 and 2) or severe IVH (stages 3 and 4). In the next step, factors with a p-value below 0.05 were entered into a multiple (multi-variable) linear mixed model and presented with 95 % confidence intervals. Simple linear mixed model analyses were performed for the central macular thickness (A1) and VA and SE within both the preterm and control groups. In all linear mixed models, the subject was the random factor.

Paper IV The means and standard deviations were calculated for the ffERG values (the amplitudes and implicit times of the six ffERG protocols). A series of linear mixed models were used to analyse and compare the ffERG values of the preterm and control groups. As in paper III, the eye (RE and LE) and group (preterm and control group) were the fixed factors and the subjects were the random factor. First, the preterm group was compared to the control group regarding the ffERG values, with an adjustment for gender and refrac-tion (SE). Then, a series of linear mixed models were used within the pre-term and control groups separately for the independent factors: VA, SE and gender, for each of the ffERG values. In the preterm group, the factors GA, BW and previous ROP (present or not, irrespective of stage or treatment) were compared to the ffERG values. Further analyses of ROP were done in the preterm group, dividing the ROP groups into: no, mild (stages 1 and 2), severe (stages 3 and 4) and severe treated ROP versus severe untreated ROP. The results from the linear mixed models were presented with 95% confi-dence intervals.

42

Results

Paper I A total of 58 children (28 girls, 30 boys) with a mean (range) age of 10.6 (6-15) years participated in examinations with SD-OCT Cirrus and TD-OCT Stratus. The left eye of one child had to be excluded because of astigmatism > 2.0 dioptres. The right eye of one child was excluded because of large eye movements distorting the OCT signal. Technical problems with the Stratus TD-OCT resulted in examinations in only 14 REs and 11 LEs. The mean (range, SD) VA of the REs was -0.043 (-0.2 - 0.1, 0.09) LogMAR and of the LEs -0.047 (-0.2 - 0.1, 0.07) LogMAR. The mean (range, SD) spherical equivalents were +0.95 (-0.5 - +2.75, 0.63) dioptres and +0.96 (+0.75 - +3.0, 0.71) dioptres. The mean OCT value of the REs and LEs are presented in Figure 19.

Figure 19. Mean (SD) macular thickness in µm of the nine ETDRS areas in the REs and LEs.

43

One eye from each child was randomised in order to present the normal val-ues (Table 2).

Table 2. Mean values of visual acuity (LogMAR), spherical equivalent (dioptres), macular thickness (µm), cube average thickness (µm) and cube volume (mm2) of the randomised eyes (n=58). The study population is divided into five age groups.

All ages (n=58)

6-7 years (n=12)

8-9 years (n=8)

10-11 years (n=12)

12-13 years (n=15)

14-15 years (n=11)

VA (SD) [Range] SE(SD) [Range] A1 (SD) [Range] A2(SD) [Range] A3 (SD) [Range] A4 (SD) [Range] A5 (SD) [Range] A6 (SD) [Range] A7 (SD) [Range] A8 (SD) [Range] A9 (SD) [Range] CV (SD) [Range] CAT (SD) [Range]

- 0.04 (0.07) -0.2 - 0.1 +0.91 (0.66) [-0.5 - +3.0] 255 (17) [218-303] 330 (16) [299-369] 316 (16) [281-353] 325 (16) [295-361] 330 (17) [300-377] 290 (15) [254-319] 273 (14) [240-303] 277 (14) [245-310] 307 (15) [275-344] 10.3 (0,5) [9.3-11.3] 289 (13) [260-315]

0.02 (0.04) [0.0 - 0.1] +1.57 (0.67) [+0.8 - +3.0] 252 (15) [218-267] 325 (15) [302-347] 309 (13) [293-331] 321 (14) [304-339] 324 (16) [302-344] 291 (14) [271-319] 269 (10) [258-290] 278 (10) [260-289] 305 (11) [285-323] 10.3 (0,4) [9.8-10.7] 288 (10) [273-299]

-0.04 (0.06) [-0.1 - 0.0] +1.09 (0.67) [+0.5 - +2.5] 255 (16) [230-275] 330 (16) [304-354] 316 (15) [293-335] 322 (17) [299-351] 332 (18) [300-358] 288 (15) [266-312] 273 (15) [254-299] 279 (18) [258-310] 308 (16) [288-337] 10,4 (0,53) [9.7-11.3] 290 (15) [270-315]

-0.05 (0.09) [-0.2 - 0.1] +0.78 (0.41) [+0.1- +1.4] 243 (11) [223-262] 323 (13) [307-352] 310 (14) [292-346] 318 (13) [307-352] 322 (14) [308-360] 281 (13) [254-299] 267 (11) [252-292] 271 (12) [255-302] 299 (14) [275-384] 10,1 (0,39) [9.5-11,1] 283 (11) [266-311]

-0.11 (0.08) [-0.2 - 0.0] +0.75 (0.57) [-0.1 - +2.1] 262 (20) [227-303] 336 (16) [312-367] 322 (17) [296-353] 331 (15) [309-361] 336 (18) [312-370] 293 (16) [272-317] 277 (16) [253-303] 279 (14) [259-309] 312 (18) [287-344] 10,5 (0,51) [9.8-11.23] 293 (14) [274-315]

-0.04 (0.11) [-0.2 - 0.1] +0.42 (0.41) [-0.5 - +1.0] 263 (18) [243-293] 335 (18) [299-369] 321 (18) [281-350] 331 (16) [295-357] 336 (19) [302-377] 292 (14) [259-310] 277 (16) [240-295] 281 (15) [245-300] 311 (14) [283-331] 10,5 (0,49) [9.3-11.1] 293 (14) [260-312]

A1=central area, A2=inner superior area, A3=inner temporal area, A4=inner inferior area, A5=inner nasal area, A6=outer superior area, A7=outer temporal area, A8=outer inferior area, A9=outer nasal area. CV=cube volume, CAT=cube average thickness, N= number, SE=spherical equivalent, SD=standard deviation, VA= visual acuity There was no significant correlation between VA or SE and the EDTRS areas (A1-A9), inner- and outer circles, CAT or CV. A significant correla-tion was found between age and the inner circle (rs=0.27, p=0.04). Male gender was associated with a thicker inner circle, mean difference: 8.7 µm (95% CI, 0.7 - 16.7, p=0.034). However, after performing a multiple linear regression analysis including age and gender, the significance disappeared. The repeatability (intra-observer variation) and the reproducibility (inter-observer variation) are presented in Tables 3A and 3B.

44

Table 3A. Repeatability of Cirrus SD-OCT assessments in all EDTRS areas in the 58 randomised eyes, expressed as the coefficient of variance with standard devia-tions and intraclass correlation with confidence intervals (lower bound [LB] and upper bound [UB]).

CoV (%) SD ICC CI (LB – UB)

A1

1.01

0.91

0.968

0.948 - 0.981

A2 0.84 0.78 0.956 0.931 - 0.973 A3 0.86 0.87 0.952 0.925 - 0.970 A4 1.20 2.85 0.953 0.925 - 0.970 A5 0.74 0.72 0.968 0.950 - 0.980 A6 1.19 0.90 0.923 0.882 - 0.953 A7 0.98 0.83 0.949 0.920 - 0.969 A8 1.15 0.88 0.929 0.890 - 0.956 A9 0.66 0.59 0.975 0.961 - 0.985 CV 0.63 0.54 0.968 0.948 - 0.981 CAT

0.62 0.52 0.970 0.961 - 0.985

CoV=coefficient of variance, SD= standard deviations (SD), ICC= intraclass correlation, CI= confidence intervals, LB=lower bound, UB= upper bound, A1=central area, A2=inner superior area,A3=inner temporal area, A4=inner inferior area, A5=inner nasal area, A6=outer superior area, A7=outer temporal area, A8=outer inferior area, A9=outer nasal area, CV=cube volume, CAT=cube average thickness Table 3B. Reproducibility of Cirrus SD-OCT assessments in all EDTRS areas in the 58 randomised eyes expressed as the coefficient of variance with standard devia-tions and intraclass correlation with confidence intervals (lower bound [LB] and upper bound [UB]).

CoV (%) SD

ICC CI (LB – UB)

A1

0.92

0.78

0.973

0.954 - 0.984

A2 1.20 0.86 0.916 0.861 - 0.950 A3 1.05 0.82 0.935 0.892 - 0.961 A4 1.23 1.49 0.857 0.769 - 0.914 A5 0.99 0.75 0.948 0.913 - 0.969 A6 1.37 1.05 0.872 0.791 - 0.923 A7 1.21 0.92 0.917 0.863 - 0.951 A8 1.18 1.02 0.904 0.840 - 0.943 A9 1.12 0.90 0.925 0.875 - 0.955 CV 0.84 0.70 0.936 0.892 - 0.963 CAT 0.84 0.68 0.939 0.896 - 0.964

CoV=coefficient of variance, SD= standard deviations (SD), ICC= intraclass correlation, CI= confidence intervals, LB=lower bound, UB= upper bound, A1=central area, A2=inner superior area,A3=inner temporal area, A4=inner inferior area, A5=inner nasal area, A6=outer superior area, A7=outer temporal area, A8=outer inferior area, A9=outer nasal area, CV=cube volume, CAT=cube average thickness

45

The OCT measurements of the Cirrus SD-OCT were significantly thicker (p<0.01) in all EDTRS areas (A1-A9) than the Stratus TD-OCT measure-ments.

Paper II A total of 49 children participated in the study, of whom 71% (35/49) partic-ipated in both paper I and paper II. One child had to be excluded due to lim-ited cooperation during the mfERG measurements. The mean (range) age of the remaining 48 children (24 girls and 24 boys) was 10.9 (5-15) years. The mfERG recordings of two REs and two LEs were excluded due to distorted signals of alternating current disturbances. Hence, 46 REs and 46 LEs of 48 children were included in the analyses. The OCT assessments of one child had to be excluded because of large eye movements during the examination The mean (range, SD) visual acuity was -0.054 (-0.2 - 0.1, 0.08) LogMAR in the REs and -0.057 (-0.2 - 0.0, 0.07) LogMAR in the LEs. The mean (range, SD) spherical equivalent was +0.95 (-0.38 - +2.50, 0.56) in the REs and +0.91 (0.0 - +3.0, 0.59) in the LEs. There were no significant differences between the REs and LEs regarding the mfERG values. Hence, the results of the REs were presented. The median (interquartile ranges (IQR)) values of the mfERG assessments in 46 REs with and ranges are presented in Table 4.

46

Table 4. Median values with interquartile ranges and ranges of the mfERG meas-urements (implicit time, amplitude and response density) in 46 REs in Rings 1-3. Table approved for reuse by Springer Nature from the published article by Molnar et al., Doc Ophthalmol. (2015;131[3]:169-76).

IQR=interquartile range, nV=nanovolts, ms=milliseconds When the mfERG measurements (implicit time, amplitude and response density) in Rings 1-3 were compared, there was a significant difference between the measurements in all rings (p < 0.001), which was most pronounced for the response density (nV/deg2) (Figure 20).

47

Figure 20. Median values of response density (nV/deg2) with interquartile ranges in Rings 1-3 with p-value from the Friedman rank sum test. Figure approved for reuse by Springer Nature from the published article by Molnar et al., Doc Ophthalmol. (2015;131[3]:169-76).

Boys had significantly longer implicit times than girls in the Rings 1-3. No gender difference was found regarding the amplitudes or response densities. Significant correlations were found between age and implicit time in Ring 1 (rs=0.42, p=0.004) and Ring 2 (rs=0.32, p=0.032). There was no significant correlation between age and the amplitude or response density. Regarding implicit time in Rings 1-3, an analysis of covariance, including gender and age, revealed that implicit time increased with age and was slightly longer in boys (Figure 21).

48

Figure 21. Scatter plot of the implicit times in ring 1 in girls and boys with increas-ing age. The solid lines show the predicted values for the girls and boys, and the dashed lines show the 95% prediction intervals. Figure approved for reuse by Springer Nature from the published article by Molnar et al., (Doc Ophthalmol. 2015;131[3]:169-76).

There was a significant correlation between implicit time in Ring 1 and the inner macular circle (rs=0.37, p=0.014).

Paper III Of the original cohort living in Stockholm and Uppsala health care regions, 67% (134/199) of the preterm children and 90% (145/162) of the control children had OCT images fulfilling the inclusion criteria in at least one eye and thereby constituted the study population. The mean (range) age of the children at examination was 6.6 (6.3-7.1) years in the preterm group (57 girls, 77 boys) and 6.5 (5.7-7.1) years in the control group (28 girls, 37 boys). The mean (range, SD) GA was 25 (23-26, 1.0) weeks and the mean (range, SD) birth weight (BW) was 803 (492-1.218, 167) g in the preterm group. Mild ROP was diagnosed in 41% (55/134), severe ROP in 32% (43/134) and 18 children were laser treated for ROP. Mild IVH was diag-nosed in 32% (43/134) and severe IVH in 6% (8/134) of the children. The mean VA and SE are presented in Table 5.

49

Table 5. Visual acuity in LogMAR and decimal acuity together with spherical equivalent in the REs and LEs of the study cohort (134 preterm children, 145 con-trols). Preterm group

(n=134) Control group (n=145)

LogMAR VA RE Mean (SD) [Range] Decimal VA RE Mean (SD) [Range]

0.11 (0.22) [-0.1 - 1.7] 0.82 (0.23) [0.02 -1.25]

0.02 (0.08) [-0.1 - 0.4] 0.97 (0.16) 0.40 - 1.25

LogMAR VA LE Mean (SD) [Range] Decimal VA LE Mean (SD) [Range]

0.09 (0.16) [-0.1 - 1.5] 0.86 (0.20) [0.03 - 1.25]

0.01 (0.08) [-0.2 - 0.2] 0.98 (0.17) [0.63 -1.60]

SE RE Mean (SD) [Range]

+1.42 (1.98) [-11.13 - +7.50]

+1.33 (0.90) [-1.00 - +6.13]

SE LE Mean (SD) [Range]

+1.34 (2.15) [-9.38 - +7.38]

+1.37 (0.84) [-0.38 - +6.25]

LE=left eye, RE=right eye, SD= standard deviation, SE=spherical equivalent VA=visual acuity

In the preterm group, adequate OCT assessments were obtained in both eyes of 107 children and in one eye (right or left) of 27 children. In the control group, 136 obtained adequate OCT assessments in both eyes, and 9 children in one eye (right or left). The mean macular thicknesses (ETDRS areas A1-A9) with standard deviations are presented in Figures 22A and 22B.

50

Figure 22A. Mean (SD) macular thickness (µm) in EDTRS areas A1-A9 in the right eyes and the left eyes in children born extremely preterm.

Figure 22B. Mean (SD) macular thickness (µm) in EDTRS areas A1-A9 in the right eyes and the left eyes in children born at term. The linear mixed model, adjusted for gender revealed a significantly thicker central macula (A1) in the preterm group than in the control group (95% confidence interval [CI]), 29.8 - 39.1; p<0.001), but there were no significant differences regarding the inner and outer circles. There was no significant association between VA or SE and central macular thickness (A1) in any of the preterm and control groups.

51

Further analyses within the preterm group revealed significant associations between the central macular thickness (A1) and GA (p=0.009), BW (p=0.037), previous ROP (p=0.002) and male gender (p=0.008). A multiple linear mixed model analysis was performed, including A1 together with GA, previous ROP and sex (male). The analysis revealed a reduction in central macular thickness (A1) by 3.94 µm per increase in gestational week (95% CI, -7.51 - -0.37; p=0.033). The presence of previous ROP (mean difference: 7.30 µm [95% CI, 1.94 - 12.67; p=0.009]) and being male (mean difference: 9.23 µm [95% CI, 2.46 - 16.01; p=0.008]) were also associated with a thick-er central macula (A1). A significant relationship between severe IVH and a thinning of the inner circle was found with a mean difference of 16.53 um (95% CI, -26.97 -6.09; p=0.002).

A significantly lower LogMAR visual acuity was found in the preterm group when compared to the control group (REs: mean difference: 0.09, 95% CI, 0.05-0.13; p<0.001, LEs: mean difference: 0.07, 95% CI, 0.04 - 0.10; p<0.001). There was no significant association between VA or SE and the central macular thickness (A1) within the preterm and control groups.

Paper IV Of the original cohort living in Uppsala health care region, 84% of the pre-term children (73/87) and 97% of the control children (64/66) participated in a 6.5-year ophthalmological follow-up within the EXPRESS study. The study population of Paper VI consisted of children obtaining ffERG assess-ments in at least one eye according to the inclusion criteria: 52 preterm chil-dren (19 girls/33 boys, 50 REs and 48 LEs) and 45 control subjects (22 girls/23 boys 45 REs, 45 LEs). The mean (range) age at examination was 6.6 (6.3-6.7) years in the preterm group and 6.6 (6.3-6.7) years in the control group. The children born preterm had a mean (range, SD) GA of 25 (23-26, 1.1) weeks and a mean (range, SD) BW of 766 (492-1218, 175) g. Mild ROP was diagnosed in 44% (23/52), severe ROP in 21% (11/52) and 6 children were treated. The mean VA and SE of the preterm and control groups are presented in Table 6.

52

Table 6. Visual acuity in LogMAR and decimal acuity together with spherical equivalent in the REs and LEs of the study cohort (52 preterm children, 45 control children).

Preterm group (n=52)

Control group (n=45)

RE

LE

RE

LE

LogMAR VA Mean (SD) [Range] Decimal VA Mean (SD) [Range]

(n=50) 0.13 (0.20) [0.00 - 0.70] (n=50) 0.78 (0.24) [0.2 - 1.0]

(n=48) 0.12 (0.31) [-0.10 - 2.00] (n=48) 0.87 (0.23) [0.01 - 1.25]

(n=45) 0.03 (0.06) [-0.10 - 0.20] (n=45) 0.94 (0.11) [0.63 - 1.25]

(n=45) 0.03 (0.07) [-0.10 - 0.2] (n=45) 0.94 (0.14) [0.63-1.25]

SE Mean (SD) [Range]

(n=50) +1.59 (2.34) [-7.63 - +7.50]

(n=48) +1.18 (3.44) [-13.25 - +7.38]

(n=44) +1.39 (0.70) [-0.25 - +4.00]

(n=44) +1.35 (0.64) [-0.25 -+3.63]

LE=left eye, n=numbers, N=numbers, VA=visual acuity, RE=right eye, SD=standard deviation, SE=spherical equivalent

The mixed model analyses revealed significantly lower LogMAR VA (mean difference: 0.082, 95% CI, 0.017 - 0.147; p=0.015) and higher values of the SE (mean difference: 0.944, 95% CI, 0.297 - 1.592; p=0.005) in the preterm group in comparison to the control children. The preterm group had signifi-cantly lower amplitudes of the a-wave of the dark-adapted 3 ERG (mean difference: -48.9µV, 95% CI, -80.0 - -17.9; p=0.003) and the dark-adapted 12 ERG (mean difference: -55.7 µV, 95% CI, -92.5- -18.8; p=0.004), togeth-er with the peak of 30 Hz flicker (mean difference: -12.1 µV, 95% CI, -22.5- -1.6; p=0.027), when compared to the control group. The preterm children had significantly prolonged implicit times of the a-wave of the dark-adapted 12 ERG (mean difference: 1.2 ms, 95% CI, 0.3 - 2.0; p=0.010) and of the peak of 30 Hz flicker ERG (mean difference: 1.2 ms, 95% CI, 0.5 - 1.8; p<0.001) in comparison to the control children. Further analyses within the preterm group revealed no significant association between GA, BW, previ-ous ROP, stage of ROP or treated ROP and the ffERG values (implicit times and amplitudes of the six ffERG protocols). Mixed-model analyses, includ-ing the ffERG values, gender, VA and SE within the preterm and the control groups, revealed only a few significant p-values indicating no systematic relationships. There was no significant difference between the six ffERG values in the REs and LEs within either the preterm or control group.

53

Discussion

Papers I and II These strictly population-based studies obtained normative data on macular thickness assessed with SD-Cirrus OCT, and also data on macular function assessed with binocular mfERG in healthy children aged 5-15 years. In paper I, the central macular thickness (A1) was the thinnest area and the inner circle the thickest area, which accords with a few previous published papers on normative data of Cirrus SD-OCT in children (Al-Haddad et al., 2014; Altemir et al., 2013; Barrio-Barrio et al., 2013) and with macular his-tology (Hendrickson, 1992; Hendrickson & Yuodelis, 1984; Vajzovic et al., 2012; Yuodelis & Hendrickson, 1986). The differences between the right and left eyes were small. There were no significant correlations between macular thickness and visual acuity, refraction (SE), age and gender. The relationship between the total macular thickness and the visual acuity in healthy children has not been investigated to a greater extent. Previous stud-ies on adults with macular oedema reported a modest correlation between visual acuity and the central macular thickness (Blumenkranz et al., 2010; Browning et al., 2007). Further, it has been described that there is no correla-tion between total central macular thickness and the visual acuity in patients with foveal hypoplasia (Holmström et al., 2010), and the absence of a foveal pit alone has not been associated with poor visual acuity (Marmor et al., 2008). In a paper by Thomas et al., however, visual acuity was associated with different grades of foveal hypoplasia where certain structural foveal features had been taken into account (Thomas et al., 2011). Regarding the refraction in paper I, all children with high refractive errors were excluded, which could have influenced the results as previous papers have reported a relationship between refractive errors and macular thickness in adults (Song et al., 2014; von Hanno et al., 2016). In the examined paediatric population aged 6-15 years in paper I, age did not have an effect on macular thickness, which is in accordance with the population-based study by Eriksson et al. (Eriksson et al., 2009). However, Barrio-Barrio et al. reported increasing age to be associated with a thicker macula in children (Barrio-Barrio et al., 2013) but the age span in that study was different which might have influenced the results. In contrast, Eriksson et al. found that macular thickness decreased with increasing age in healthy adults (Eriksson & Alm, 2009; Song et al.,

54

2010). We believe that age does not have to be taken into consideration when examining children between 6-15 years of age with Cirrus SD-OCT. Boys had a slightly thicker macula than girls as reported by Barrio-Barrio et al. (Barrio-Barrio et al., 2013) but after performing a multiple linear regres-sion analysis the significance disappeared. A gender difference in adults, however, where men have thicker maculae than women, has been reported (von Hanno et al., 2016). The repeatability, reproducibility and intraclass correlations of the SD-Cirrus OCT measurements in the paediatric popula-tion were good and in agreement with Altermir et al. (Altemir et al., 2013) and Garcia Martin et al. (Garcia-Martin et al., 2011). Hence, in a clinical situation, one examination performed by different examiners with Cirrus SD-OCT is considered reliable. The macular Cirrus SD-OCT assessments were approximately 30% thicker than the Stratus TD-OCT measurements due to a different segmentation algorithm (Mylonas et al., 2009) and the macular thickness assessments were not interchangeable. It is, therefore, important that only one of the OCT-method should be chosen when monitor-ing a patient. In paper II, assessing the macular function with mfERG, only the results of the right eyes were reported and presented since the differences between the right and left eyes were small. The implicit time, amplitude and response density varied significantly between Rings 1, 2 and 3, and the variation was most pronounced for response density in accordance with previous papers (Fulton et al., 2005; Jackson et al., 2002; Michalczuk et al., 2016; Verdon & Haegerstrom-Portnoy, 1998; Åkerblom et al., 2016). This finding revealed that the fixation must have been good since the density of cone photorecep-tors is highest in the fovea (Curcio et al., 1990) and the highest mfERG re-sponse is expected in the central area (Ring 1) if the fixation is adequate. The implicit time in Rings 1 and 2 increased slightly with age and was slightly longer in boys in Rings 1-3. In adults, the implicit time increases slowly with age (Gerth et al., 2003; Jackson et al., 2002; Seiple et al., 2003), but to our knowledge it has not been reported in children before. The boys had slightly longer implicit times in than girls, which is in line with mfERG-studies in adults (Ozawa et al., 2014). Gender differences in the electrophysiological response assessed with ffERG (Birch & Anderson, 1992) and VEP have been reported (Dion et al., 2013). One could speculate whether sex hor-mones affect the retinal function, since the presence of sex hormone recep-tors in the retina has been reported (Munaut et al., 2001). Finally, in the healthy paediatric cohort in paper II, there was a relationship between macu-lar function and structure: the implicit time in Ring 1 became longer with increasing thickness of the macular inner circle. The implicit time has been described as prolonged in conditions with macular pathology, such as in patients with diabetic retinopathy where increasing macular thickness of macular oedema results in longer implicit times (Holm et al., 2007).

55

Papers III and IV These population-based studies investigated the macular thickness with Cir-rus SD-OCT and the retinal function with binocular ffERG in 6.5-year-old children born extremely preterm and in a control group born at term − all of whom were participating in the EXPRESS study (EXPRESS Group, 2010). The children born EPT had a significantly thicker central macula (A1) than the children born at term, which accords with previous studies (Bowl, Stieger, et al., 2016; Ecsedy et al., 2007; Rosén et al., 2015; Tariq et al., 2012; Wang et al., 2012; Wu et al., 2012; Åkerblom et al., 2011). The EPT group had a lower visual acuity than the control group. No significant asso-ciation was found between the visual acuity and the central macular thick-ness in either group, which is in accordance with previous papers on children born preterm (Bowl, Lorenz, et al., 2016; Tariq et al., 2012; Wang et al., 2012; Wu et al., 2012; Åkerblom et al., 2011) and also with the findings in paper I. Further investigation within the EPT group revealed low GA, former ROP and male gender as important factors for a thicker central macula. According to the multiple linear mixed model, including all those factors the central macular thickness was reduced by 3.94 µm per additional gestational week. Previous studies have reported the relationship between low GA and a thick-er central macula (Bowl, Lorenz, et al., 2016; Wang et al., 2012; Wu et al., 2012; Åkerblom et al., 2011). Wang et al found a steep decrease of 14.3 µm per additional gestational week in children born before 28 weeks of GA. However, the number of extremely preterm children in that study was small-er and no adjustment for gender had been performed, which could explain the different findings. The finding that GA has an influence on the central macular thickness is in accordance with morphological studies describing important steps of the macular development at 24-25 weeks of GA, when the foveal pit appears and the avascular zone is created (Hendrickson et al., 2012; Hendrickson & Yuodelis, 1984; Provis & Hendrickson, 2008). In pa-per III, former ROP in the EPT group was associated with a thicker central macula, though the reason for ROP affecting macular thickness is not fully known. However, macular abnormalities have been associated with ROP such as macular heterotopia (Soong et al., 2008), and idiopathic maculopathy (Lee et al., 2010). Chavala et al. described the presence of preretinal macular structures close to blood vessels in preterm infants with advanced ROP ex-amined with handheld OCT (Chavala et al., 2009), which highlights the rela-tionship between ROP and macular structure. Previous studies have reported different conclusions regarding the effect of ROP on central macular thick-ness in preterm children. According to Escedy et al. ROP was the main risk factor in contrast to Åkerblom et al. and Bowl et al., where GA was the only

56

risk factor after multiple regression analyses (Bowl, Lorenz, et al., 2016; Ecsedy et al., 2007; Åkerblom et al., 2011). In a study by Wu et al., children who had been laser treated or cryo-treated for ROP had a thicker fovea in comparison to preterm children without treatment or with no ROP. However, the studies are not exactly comparable since the number of subjects, age at examination, subgrouping and the range of GA of the preterm children were different. In paper III, male gender had an influence on the central macular thickness as discussed in paper I. To our knowledge, the importance of gen-der influence on the macular thickness in children born EPT has not been reported before. Finally, there was an association between a thinner inner circle and severe IVH in the EPT group. One explanation for this could be a possible loss of ganglion cells due to retrograde neuronal degeneration in the optic pathways caused by IVH. A relationship between damage to the optical radiation and reduced retinal nerve fibre thickness has recently been report-ed, where retrograde degeneration of the retinal neurons was suggested as a cause (Lennartsson et al., 2014). In paper IV, assessing the retinal function with ffERG, the children born EPT had lower amplitudes of the a-wave of combined rod-cone response (the dark-adapted 3 and 12 ffERG) as well as of the peak of the isolated cone response (30 Hz flicker ffERG), when compared to children born at term. The implicit time was prolonged in the a-wave of combined rod and cone response (the dark-adapted 12 ffERG) and of the peak of the cone response (30 Hz flicker ffERG) in the EPT group in comparison to the control group. Since the a-wave is considered to originate from the photoreceptors (Brown & Watanabe, 1962; Brown & Wiesel, 1961) and the b-wave from bipolar cells (Robson & Frishman, 1998; Stockton & Slaughter, 1989), our findings revealed a dysfunction of the rod and cone photoreceptors as well as of the cone bipolar cells in the EPT group. Several ffERG studies have reported a retinal dysfunction in infants and children born preterm; however the majori-ty of these studies described mainly a rod dysfunction (Ecsedy et al., 2011; Fulton et al., 2008; Fulton et al., 2009; Fulton et al., 2001; Hamilton et al., 2008; Harris et al., 2011; Zhou et al., 2010; Åkerblom et al., 2014). In paper IV, the isolated cone function was significantly reduced, which might be explained by a persistent immaturity of the cone photoreceptors and bipolar cells at 6.5 years of age due to the extreme prematurity. When the extremely premature infant is born, as early as by 22 weeks of GA, most cell types can be recognised (Hendrickson & Drucker, 1992), but the retinal vascularisation is not complete (Hughes et al., 2000; Provis, 2001) and important steps of retinal development occur between Fwks 25 and 38. During this gestational period, the photoreceptors elongate, the avascular zone is created and the retinal vasculature, together with the outer plexiform layer, extend to the peripheral retina (Hendrickson, 2016; Hendrickson et al., 2012; Hughes et al., 2000; Provis, 2001; Provis & Hendrickson, 2008). The cones develop

57

before the rods and they have been observed early during the gestational period (Hendrickson, 2016; Mann, 1964; Young, 1985). Hence, it is possible that there is a persistent abnormal retinal development in both rods and cones in the child born EPT at 6.5 years of age. Analyses within the EPT group revealed no association between the ffERG-assessments and GA, ROP (irrespective of stage) or laser-treated ROP. Dif-ferent conclusions regarding factors influencing the ERG response in chil-dren born preterm have been reported (Ecsedy et al., 2011; Fulton et al., 2008; Fulton et al., 2009; Fulton et al., 2001; Hamilton et al., 2008; Harris et al., 2011). As mentioned in paper III, these studies are not comparable since the number of preterm children, the age at examination, the GA age-span and ERG methods were different. A positive correlation between the com-bined rod and cone response and increasing GA was found by Åkerblom et al (Åkerblom et al., 2011). ROP has been associated with a reduced rod function (Fulton et al., 2009; Fulton et al., 2001; Hamilton et al., 2008; Harris et al., 2011; Åkerblom et al., 2014). Both rod and cone dysfunction have been described in infants with treated ROP (Hamilton et al., 2008). In paper IV, the lack of association between the retinal dysfunction and GA or ROP might be explained by the homogenous cohort of children who were born within a very narrow GA span and had ROP diagnosed to a large extent (62%). Only six children had been laser-treated for ROP. The EPT group had a significantly lower VA and higher values of refractive errors than the control group, which is in accordance with previous studies (Holmström & Larsson, 2008; O'Connor et al., 2002). An adjustment for refractive errors and gender was made when the ffERG assessments were compared with the EPT and the control groups, since high refractive errors can affect the ERG response as well as the gender (Birch & Anderson, 1992; Flitcroft et al., 2005; Fulton & Hansen, 1995). There was no significant association between the ffERG assessments and visual acuity, refraction (SE) or gender in either the EPT or control groups.

58

Conclusion

Normative data of the OCT and binocular mfERG assessments were report-ed and the methods were well tolerated by healthy children aged 5-15 years. The cooperation was good and no child was excluded from the study popula-tion in paper I and only one child in paper II. These painless and fast exami-nations could be easily performed in children from 5 years of age without using general anesthesia. The methods provided reliable results and can be recommended for clinical practice when investigating children with different ophthalmological problems such as reduced visual acuity, nystagmus, pho-tophobia and affected colour vision. The structure and function of the macu-la are affected by several retinal diseases in childhood for example Stargardt disease, achromatopsia and X-linked juvenile retinoschisis (Eksandh et al., 2002; Eriksson et al., 2004; Fujinami et al., 2015; Kjellström et al., 2010). As the OCT technology continuously evolves, further evaluation of the gan-glion cell layer, the photoreceptor layer (Liu et al., 2016), choroidal thick-ness (Yokouchi et al., 2016) and retinal vasculature (Koustenis et al., 2017) is possible today, which could add even more information about the retina and improve diagnostics. Children born EPT aged 6.5 years had a significantly thicker central macula and both rod and cone function was significantly reduced in comparison to children born at term. A relationship between GA, male gender and ROP and central macular thickness was found. These findings suggest that there is an abnormal retinal development with a persistent retinal immaturity in children born EPT. Symptoms such as nyctalopia, visual field loss, reduced colour vision and photophobia are well known in rod and cone disorders (Gränse et al., 2004; Michaelides et al., 2004). One can only speculate how abnormal retinal development will be expressed in these children. Long-term follow up studies are therefore necessary in order to evaluate the functional ophthalmo-logical outcome in this vulnerable population of children growing up today.

59

Sammanfattning på svenska

Tidig diagnos hos barn med sjukdom i näthinnan är extremt viktig. Näthin-nesjukdomar kan ge upphov till symtom som synnedsättning, ljuskänslighet, påverkat färgsinne, nedsatt mörkerseende och inskränkta synfält. En mängd olika sjukdomar kan drabba näthinnan och dess funktion i olika åldrar som exempelvis Stargardt sjukdom, akromatopsi och X-bunden retinoschis. En tidig diagnos möjliggör korrekt synhabilitering och rådgivning till familj, barnomsorg och skola. I framtiden kan även genbehandling av näthinnesjuk-domar bli möjligt. Då är det av stor vikt att kunna identifiera barn som kan vara aktuella för just denna behandling och för rätt genetisk rådgivning. För att kunna ställa rätt diagnos är undersökningsmetoder med optical coherence tomography (OCT), multifokal electroretinografi (mfERG) och fullfälts el-ektroretinografi (ffERG) av stort värde. OCT-metoden avbildar näthinnan med hjälp av reflektion av nära infrarött ljus. Med hjälp av denna metod kan man mäta näthinnetjockleken samt få en bild av näthinnans struktur som vid vissa näthinnesjukdomar har ett speciellt strukturellt utseende och en viss näthinnetjocklek. OCT-tekniken är speciellt barnvänlig då mätningen går mycket fort att genomföra och endast kräver att patienten tittar mot ett fixationsobjekt i apparaten (Figur 23). Denna metod går att utföra från c:a 4 års ålder på barn. Funktionen av näthinnans syncel-ler, dvs. stavar och tappar, kan mätas med ffERG och mfERG. FfERG är en metod för undersökning av stavarnas och tapparnas funktion i hela näthin-nan. Stavarna är mycket känsliga för ljus och används vid mörkerseende. Stavarna finns i hela näthinnan, men inte i den mest centrala delen av gula fläcken. Tapparna är inte lika ljuskänsliga och används vid dag- och färgse-ende. I den mest centrala delen av gula fläcken (makula) finns endast tappar, i övrigt finns de utspridda över hela näthinnan. Då ögat stimuleras med olika typer av ljus uppstår små, elektriska potentialer från näthinnan. Med hjälp av elektroder som placeras i ögat kan dessa små elektriska utslag mätas. Elektroretinografi återspeglar därmed näthinnans funktion och ger en objek-tiv bedömning av synfunktionen. MfERG är en objektiv funktionsmätning av i huvudsak tapparna i makula. Makula är ett viktigt område i näthinnan som ger den maximala synskärpan och vårt färgseende. Både ffERG och mERG är barnvänliga metoder som kan utföras på barn från 5-6 års ålder utan sövning. Normalvärden av OCT-, ffERG- samt mfERG hos friska barn är en förutsättning för att kunna utvärdera om det förekommer näthinnesjuk-

60

dom hos barn med exempelvis oklar synnedsättning. Sådana normalvärden saknades för OCT- och mfERG mätningar med apparaterna av typen Cirrus och Espion. Avancerad högteknologisk sjukvård och nya behandlingsmetoder har med-fört ökad överlevnad av för tidigt födda barn. Denna utveckling har dock varit associerad med en ökning av allvarliga sjukdomar under nyföddhetspe-rioden som exempelvis omogenhetssjukdom i näthinnan, s.k. prematuritets-retinopati (retinopathy of prematurity (ROP)), omognad av lungorna, hjärn-blödning och inflammatorisk tarmsjukdom. Det mest avancerade stadiet av ROP kan leda till avlossning av näthinnan med blindhet som följd. Risken för ROP och fram för allt de allvarligare graderna av sjukdomen ökar ju lägre mognadsgrad och vikt barnet har vid födseln. Idag är den gängse be-handlingsmetoden för allvarligare stadier av ROP laserbehandling av näthin-nan. Näthinnans utveckling är mycket komplex och startar tidigt under fostersta-diet. Cellerna bildas i en tidsbestämd ordning där tapparna bildas före sta-varna. Vid mitten av graviditeten finns näthinnans alla celler representerade men de är fortfarande omogna. Utvecklingen av näthinnans celler fortsätter flera år efter födseln. Viktiga steg i näthinnans utveckling äger rum vid tiden för det extremt prematura barnets födelse och dessa barn föds med en mer omogen näthinna än ett barn fött i normal tid. Histologiska- samt OCT-studier av näthinnan har hos barn, födda i normal tid, visat att den först vid 13-16 års ålder har ett utseende likt den vuxnes. Stora populationsbaserade uppföljningsstudier av för tidigt födda barn har visat att det föreligger en ökad risk för ett flertal oftalmologiska problem senare under barndomsperioden såsom nedsatt syn, skelningar, brytningsfel, nedsatt kontrastseende, påverkat synfält samt visuella perceptionsproblem jämfört med barn födda i normal tid. Flera studier har också visat att prema-turfödda barn har strukturella avvikelser i näthinnan och ett begränsat antal elektrofysiologiska studier har rapporterat försämrad näthinnefunktion under nyföddhetsperioden och under barndomen. I Sverige pågår en nationell stu-die, EXPRESS-studien som inkluderade samtliga barn födda före gravidi-tetsvecka 27 under åren 2004-2007. I nyföddhetsperioden hade dessa barn en hög förekomst av ROP som var behandlingskrävande. Vid 2.5-års uppfölj-ningen rapporterades det att 33% av de prematurfödda barnen hade syn-funktionsproblem som synnedsättning, brytningsfel eller skelning. En 6.5-års uppföljning visade att en ännu högre andel av de tidigt födda barnen hade ögonproblem; 38% hade låg synskärpa, höga brytningsfel samt skelning. I arbete I och II ingick 200 barn mellan 5-15 år, slumpmässigt utvalda från Socialstyrelsens födelseregister med inklusionskriterierna: födsel ≥ 37 veck-

61

or, födelsevikt ≥ 2500 g och boende i Uppsalaregionen. Föräldrar till barnen fick lämna ett skriftligt godkännande och barnet fick lämna ett muntligt god-kännande till deltagande. Inklusionskriterier för studien var att vara tidigare frisk, en synskärpa på vardera öga på minst 0.8 decimal synskärpa (0.1 LogMAR) och ingen förekomst av höga brytningsfel. I arbete I utfördes OCT-undersökningar av makula med apparaten Cirrus och den äldre typen av OCT, s.k. Stratus. A.M. utförde tre Cirrus OCT under-sökningar i följd och en forskningssjuksköterska E.N. tre stycken därefter. Slutligen undersökte E.N. barnen med tre Stratus OCT-undersökningar. Femtio-åtta barn medverkade totalt i studien. Resultaten visade att det inte fanns systematiska skillnader i OCT-mätningarna mellan upprepade under-sökningar av en och samma undersökare samt mellan två olika undersökare. Det fanns inte heller någon systematisk skillnad mellan mätningar av höger och vänster ögon. Det påvisades ingen relation mellan makulatjockleken och ålder samt kön i de slutgiltiga analyserna. Cirrus OCT gav högre tjockleks-värden i makula än Stratus OCT vilket berodde på att beräkningen av tjock-leken skedde på olika sätt i apparaterna. Mätningar med samma apparat är därför nödvändig vid uppföljning av en patient. Sammanfattningsvis bedöm-des Cirrus OCT-mätningar av makula som tillförlitliga och studien ledde till ett användbart normalmaterial på barn i åldrarna 5-15 år. I arbete II utfördes mfERG-undersökningar med tunna trådelektroder som placerades innanför det nedre ögonlocket efter droppbedövning och med apparaten Espion. I denna studie accepterade 49 barn att delta och totalt kunde mfERG-mätningar på 48 barn inkluderas i beräkningarna. Det fanns ingen systematisk skillnad vad gäller mätningarna i höger och vänster öga. MfERG-mätningarnas utslag överensstämde med hur tätt syncellerna är be-lägna i makula. Pojkar hade längre överledningstid än flickor. Sammanfatt-ningsvis bedömdes denna mfERG-undersökningsmetod som tillförlitlig och passande till kartläggning av makulas funktion. Studien resulterade i ett an-vändbart normalmaterial för mfERG-undersökningar av barn.

I arbete III undersöktes 6.5 år gamla barn födda extremt tidigt samt en kon-trollgrupp av barn födda i normal tid; alla medverkande i EXPRESS-studien och var boende i Uppsala-Stockholmregionen. OCT-undersökningar med apparaten Cirrus från 134 barn födda extremt tidigt och från 145 kontroll-barn kunde analyseras. Barnen födda extremt tidigt hade en tjockare central makula än kontrollbarnen. Mognadsålder vid födelse, förekomst av ROP (oavsett grad och behandling) och manligt kön var faktorer som tillsammans inverkade på makulatjockleken. Vid varje veckas minskning i mognadsålder så ökade makulatjockleken i snitt med 3.94 µm. Det fanns inget samband mellan synskärpan och den totala tjockleken i makula i prematur- och kon-trollgruppen.

62

I arbete IV undersöktes 6.5 år gamla barn födda extremt tidigt respektive en kontrollgrupp födda i normal tid; alla medverkade i EXPRESS-studien och boende i Uppsalaregionen. FfERG-undersökningar utfördes med trådelek-troder. Undersökningar från 52 barn födda extremt tidigt och 45 kontrollbarn ingick i analyserna. De barn som var födda extremt tidigt hade lägre utslag av de elektriska potentialerna i de kombinerade stav-och tappsvaren men även när det gällde det renodlade tappsvaret i jämförelse med kontrollbar-nen. Överledningstiden var också förlängd i det kombinerat stav-tappsvaret samt i det renodlade tappsvaret. Inom prematurgruppen fanns det inget sam-band mellan mognadsålder eller ROP (oavsett grad eller om behandling fö-rekommit) och ffERG-mätningarna. Sammanfattningsvis har arbete I och II visat att OCT-undersökningar av makula med apparaten Cirrus och mfERG-undersökning med apparaten Espion med trådelektroder är tillförlitliga metoder som är passande för barn. Arbete III och IV har visat att extremt tidig födsel påverkar strukturen i cen-trala makula samt näthinnans funktion vid 6.5 års ålder. Arbete IV påvisades en nedsatt stav- och tapp funktion men även en nedsatt renodlad tappfunkt-ion hos barn födda extremt tidigt. Tidigare studier har visat att det framför allt är stavfunktionen som är påverkad av tidig födsel. Mognadsålder, ROP och manligt kön var faktorer som tillsammans inverkade på makulatjockle-ken hos de extremt tidigt födda. I arbete IV inverkade inte mognadsålder, ROP eller kön på ffERG-mätningarna. Studierna visade att extrem prematuritet gav en kvarvarande strukturell av-vikelse i centrala makula samt en nedsatt funktion av stavar och tappar samt enbart av tappar vid 6.5 års ålder. ROP hade en inverkan på retinas centrala struktur men inte på funktionen enligt arbete III och IV. En nedsatt funktion av stavar och tappar vid näthinnesjukdomar kan ge symtom som nedsatt mörkerseende, bländningsbesvär och påverkat färgseende. Man kan enbart spekulera i vad dessa avvikelser kan leda till för syn- och ögon symtom hos denna grupp av barn. Uppföljningsstudier är därför av största vikt för att korrekta oftalmologiska uppföljningsrutiner samt habilitering och stöd i sko-lan skall kunna ges. I nyföddhetsperioden får dessa barn en mycket avance-rad och högteknologisk vård under flera månader. Under uppväxten är det också viktigt att de får möjlighet till samma noggranna omhändertagande då det dagliga livet ställer höga krav på synfunktionen.

63

Figur 23. Ett barn genomgår en OCT-undersökning av makula med apparaten Cirrus av A.M (bilden publicerad med tillstånd av barn och vårdnadshavare).

64

Acknowledgements

I owe my deepest gratitude to Uppsala University and to all the people that have been involved in this thesis. Without you it would not have been possi-ble. I would like to express my sincere appreciation and thanks especially to: Professor Gerd Holmström, my main supervisor, – a wonderful mentor and teacher. Thank you for all the effort and energy you put into me, for all your support and wise advice. Thank you for always believing in me and being a true inspiration and role model for research and paediatric ophthalmology. Whenever I needed help, you reached out to me without hesitation. I have grown as a researcher and clinician because of you. Associate Professor Eva Larsson, my co-supervisor, – a passionate and skilled researcher and clinician. Thank you for all the wise advice, support, your great and instant feedback and for always being so encouraging. You have an ability to see the bright side of most things, which has made all the work – which sometimes has been hard – so much easier. Thank you for excellent scientific guidance. You are brilliant. Professor Sten Andréasson, my co-supervisor, – a great researcher and ex-perienced clinician. You have truly inspired me into the field of electrophys-iology. I appreciate our interesting and inspiring scientific discussions. Thank you for your enthusiasm and support. I truly admire your knowledge and your personality.

Professor Per Söderberg, Chair of Ophthalmology, Dept. of Neuroscience. Thank you for all the valuable scientific discussions regarding methods and statistical analyses. The journal clubs have been very inspiring to me. You have helped me to grow as a PhD student and taught me to analyse scientific papers in a very critical way.

Head of the Ophthalmological Department, Uppsala University Hospital Manochehr Amani, for giving me the opportunity to start my PhD studies

65

during my residency in ophthalmology. Thank you for always being so sup-portive. Hanna Åkerblom, M.D., PhD, co-author, for being so enthusiastic and en-couraging. Thank you for all your help with the electrophysiological exami-nations and great feedback on the papers. Kerstin Hellgren (M.D. PhD), Maria Nilsson, (senior lecturer, PhD) and Rebecka Rosén (optician, PhD student), my co-authors. It was a true pleas-ure working with all of you. Our excellent cooperation has yielded great results. The EXPRESS group, for the possibility of examining the subcohort of children in the EXPRESS study. Research nurse Eva Nuija, for all evenings working together with the study participants. Thank you for all your wonderful and efficient help in all my papers. I enjoyed every minute working with you. You are the perfect re-search nurse.

Orthoptists Jonina Hreinsdottir and Lena Falkman, who participated in the EXPRESS study and performed the ophthalmological examinations. Biomedical analysts Margareta Grindlund and Berit Lindgren for the fantastic help with the electrophysiological examinations.

Senior Research Engineer Olav Mäepea, who without hesitation helped me with a great numbers of figures and pictures for my papers and this thesis. Even though you were very busy, you always took the time for me in a very enthusiastic manner.

All study participants and their parents. Thank you for your time and energy. It has meant a lot to me! Statisticians Marcus Thuresson and Åsa Vernby for their valuable help with the statistical analyses. Thank you for your brilliant work. Course administrator, Dept. of Neuroscience, Gunneli Ekberg, for always being so helpful with the administrative tasks regarding my PhD studies. Thank you Richard Ekberg for your participation. Clinical Assistant, Dept. of Ophthalmology, Uppsala University Hospital, Marianne Bergström, for helping me with everything from problems with the printer to planning my research periods.

66

Proofreader Keith Roberts for your great help with the formal British Eng-lish in the seemingly never-ending documents of scientific texts. Medical illustrator Kari C. Toverud for the beautiful figures in my thesis.

Photographer Sevim Yildiz, for the valuable help with figures in Photoshop. All my colleagues and the whole staff at the Ophthalmological Department at Uppsala University Hospital. Thank you all for being so encouraging and supportive! All my colleagues and the whole staff at the Ophthalmological Department at Skaraborg Hospital in Skövde, where I first started my ophthalmological career. Christina and Imrich Molnar, for being wonderful parents and giving me endless love and support! Dear father, thank you for introducing me to the beautiful world of ophthalmology. Eva Molnar, Gustav Molnar and David Molnar, for being angels in my life. Ellen Börjesson, beloved grandmother, for always believing in me and be-ing encouraging on my scientific path.

Marianne and Lennart Söderkvist, for always being so supportive. My beloved friends, thank you for always being so understanding and won-derful. I’m so grateful that you are a part of my life.

67

I gratefully acknowledge the financial support and generosity of the follow-ing foundations supporting the thesis:

Crown Princess Margareta Foundation for the Visually Impaired

Carmen & Bertil Regnér foundation

Her Majesty the Queen Silvia’s Jubilee Fund

Sigvard & Marianne Bernadotte Research Foundation

Swedish Society of Medicine

Ögonfonden

68

References

Aiello, L. P., Northrup, J. M., Keyt, B. A., Takagi, H., & Iwamoto, M. A. (1995). Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol, 113(12), 1538-1544.

Al-Haddad, C., Antonios, R., Tamim, H., & Noureddin, B. (2014). Interocular symmetry in retinal and optic nerve parameters in children as measured by spectral domain optical coherence tomography. Br J Ophthalmol, 98(4), 502-506.

Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J., & Keshet, E. (1995). Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med, 1(10), 1024-1028.

Altemir, I., Pueyo, V., Elia, N., Polo, V., Larrosa, J. M., & Oros, D. (2013). Reproducibility of optical coherence tomography measurements in children. Am J Ophthalmol, 155(1), 171-176 e171.

Andréasson, S., & Ponjavic, V. (1996). Full-field electroretinograms in infants with hereditary tapetoretinal degeneration. Acta Ophthalmol Scand Suppl(219), 19-21.

Andressen, C., Briese, V., & Ulfig, N. (2003). Progenitor Cells from Human Embryonic Retina – Proliferation and Preferential Differentiation into Ganglion Cells. Neuroembryology and Aging, 2(3), 123-129.

Austeng, D., Källén, K. B., Ewald, U. W., Jakobsson, P. G., & Holmström, G. E. (2009). Incidence of retinopathy of prematurity in infants born before 27 weeks' gestation in Sweden. Arch Ophthalmol, 127(10), 1315-1319.

Austeng, D., Källén, K. B., Ewald, U. W., Wallin, A., & Holmström, G. E. (2010). Treatment for retinopathy of prematurity in infants born before 27 weeks of gestation in Sweden. Br J Ophthalmol, 94(9), 1136-1139.

Azria, E., Kayem, G., Langer, B., Marchand-Martin, L., Marret, S., Fresson, J., . . . Ancel, P. Y. (2016). Neonatal Mortality and Long-Term Outcome of Infants Born between 27 and 32 Weeks of Gestational Age in Breech Presentation: The EPIPAGE Cohort Study. PLoS One, 11(1), e0145768.

Balasubramanian, R., & Gan, L. (2014). Development of Retinal Amacrine Cells and Their Dendritic Stratification. Curr Ophthalmol Rep, 2(3), 100-106.

Barrio-Barrio, J., Noval, S., Galdos, M., Ruiz-Canela, M., Bonet, E., Capote, M., & Lopez, M. (2013). Multicenter Spanish study of spectral-domain optical coherence tomography in normal children. Acta Ophthalmol, 91(1), e56-63.

Baylor, D. A. (1987). Photoreceptor signals and vision. Proctor lecture. Invest Ophthalmol Vis Sci, 28(1), 34-49.

Baylor, D. A., Lamb, T. D., & Yau, K. W. (1979). Responses of retinal rods to single photons. J Physiol, 288, 613-634.

Beatty, S., Koh, H., Phil, M., Henson, D., & Boulton, M. (2000). The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol, 45(2), 115-134.

69

Birch, D. G., & Anderson, J. L. (1992). Standardized full-field electroretinography. Normal values and their variation with age. Arch Ophthalmol, 110(11), 1571-1576.

Björkerud, S. r. (1971). Histologi. Stockholm,: Almqvist och Wiksell. Blumenkranz, M. S., Haller, J. A., Kuppermann, B. D., Williams, G. A., Ip, M.,

Davis, M., . . . Whitcup, S. M. (2010). Correlation of visual acuity and macular thickness measured by optical coherence tomography in patients with persistent macular edema. Retina, 30(7), 1090-1094.

Bone, R. A., Landrum, J. T., Fernandez, L., & Tarsis, S. L. (1988). Analysis of the macular pigment by HPLC: retinal distribution and age study. Invest Ophthalmol Vis Sci, 29(6), 843-849.

Bowl, W., Lorenz, B., Stieger, K., Schweinfurth, S., Holve, K., Friedburg, C., & Andrassi-Darida, M. (2016). Correlation of central visual function and ROP risk factors in prematures with and without acute ROP at the age of 6-13 years: the Giessen long-term ROP study. Br J Ophthalmol, 100(9), 1238-1244.

Bowl, W., Stieger, K., Bokun, M., Schweinfurth, S., Holve, K., Andrassi-Darida, M., & Lorenz, B. (2016). OCT-Based Macular Structure-Function Correlation in Dependence on Birth Weight and Gestational Age-the Giessen Long-Term ROP Study. Invest Ophthalmol Vis Sci, 57(9), OCT235-241.

Bradshaw, K., Hansen, R., & Fulton, A. (2004). Comparison of ERGs recorded with skin and corneal-contact electrodes in normal children and adults. Doc Ophthalmol, 109(1), 43-55.

Brown, K. T., & Watanabe, K. (1962). Isolation and identification of a receptor potential from the pure cone fovea of the monkey retina. Nature, 193, 958 passim.

Brown, K. T., & Wiesel, T. N. (1961). Localization of origins of electroretinogram components by intraretinal recording in the intact cat eye. J Physiol, 158, 257-280.

Browning, D. J., Glassman, A. R., Aiello, L. P., Beck, R. W., Brown, D. M., Fong, D. S., . . . Miskala, P. H. (2007). Relationship between optical coherence tomography-measured central retinal thickness and visual acuity in diabetic macular edema. Ophthalmology, 114(3), 525-536.

Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., . . . Nagy, A. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 380(6573), 435-439.

Cepko, C. L., Austin, C. P., Yang, X., Alexiades, M., & Ezzeddine, D. (1996). Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U S A, 93(2), 589-595.

Chan-Ling, T., Gock, B., & Stone, J. (1995). The effect of oxygen on vasoformative cell division. Evidence that 'physiological hypoxia' is the stimulus for normal retinal vasculogenesis. Invest Ophthalmol Vis Sci, 36(7), 1201-1214.

Chan-Ling, T., Tout, S., Hollander, H., & Stone, J. (1992). Vascular changes and their mechanisms in the feline model of retinopathy of prematurity. Invest Ophthalmol Vis Sci, 33(7), 2128-2147.

Chauhan DS, M. J. (1999). The interpretation of optical coherence to-mography images of the retina. Invest Ophthalmol Vis Sci, 40(10), 2332-2342.

Chavala, S. H., Farsiu, S., Maldonado, R., Wallace, D. K., Freedman, S. F., & Toth, C. A. (2009). Insights into advanced retinopathy of prematurity using handheld spectral domain optical coherence tomography imaging. Ophthalmology, 116(12), 2448-2456.

Chen, E. (1993). Refractive indices of the rat retinal layers. Ophthalmic Res, 25(1), 65-68.

70

Chen, E., Söderberg, P. G., & Lindström, B. (1991). Lipid and protein density in the rat retina: a microradiographical study. Ophthalmic Res, 23(4), 220-224.

Curcio, C. A., & Allen, K. A. (1990). Topography of ganglion cells in human retina. J Comp Neurol, 300(1), 5-25.

Curcio, C. A., Sloan, K. R., Kalina, R. E., & Hendrickson, A. E. (1990). Human photoreceptor topography. J Comp Neurol, 292(4), 497-523.

Dawson, W. W., Trick, G. L., & Litzkow, C. A. (1979). Improved electrode for electroretinography. Invest Ophthalmol Vis Sci, 18(9), 988-991.

de Kleine, M. J., den Ouden, A. L., Kollee, L. A., Ilsen, A., van Wassenaer, A. G., Brand, R., & Verloove-Vanhorick, S. P. (2007). Lower mortality but higher neonatal morbidity over a decade in very preterm infants. Paediatr Perinat Epidemiol, 21(1), 15-25.

Delori, F. C., Goger, D. G., Hammond, B. R., Snodderly, D. M., & Burns, S. A. (2001). Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry. J Opt Soc Am A Opt Image Sci Vis, 18(6), 1212-1230.

Dion, L. A., Muckle, G., Bastien, C., Jacobson, S. W., Jacobson, J. L., & Saint-Amour, D. (2013). Sex differences in visual evoked potentials in school-age children: what is the evidence beyond the checkerboard? Int J Psychophysiol, 88(2), 136-142.

Early Treatment Diabetic Retinopathy Study Research Group. (1985). Photocoagulation for diabetic macular edema. Early Treatment Diabetic Retinopathy Study report number 1. . Arch Ophthalmol, 103(12), 1796-1806.

Early Treatment For Retinopathy of Prematurity Cooperative, G. (2003). Revised indications for the treatment of retinopathy of prematurity: results of the early treatment for retinopathy of prematurity randomized trial. Arch Ophthalmol, 121(12), 1684-1694.

Ecsedy, M., Szamosi, A., Karko, C., Zubovics, L., Varsanyi, B., Nemeth, J., & Recsan, Z. (2007). A comparison of macular structure imaged by optical coherence tomography in preterm and full-term children. Invest Ophthalmol Vis Sci, 48(11), 5207-5211.

Ecsedy, M., Varsanyi, B., Szigeti, A., Szrnka, G., Nemeth, J., & Recsan, Z. (2011). Cone function in children with a history of preterm birth. Doc Ophthalmol, 122(3), 141-148.

Eksandh, L., Kohl, S., & Wissinger, B. (2002). Clinical features of achromatopsia in Swedish patients with defined genotypes. Ophthalmic Genet, 23(2), 109-120.

Eriksson, U., & Alm, A. (2009). Macular thickness decreases with age in normal eyes: a study on the macular thickness map protocol in the Stratus OCT. Br J Ophthalmol, 93(11), 1448-1452.

Eriksson, U., Holmström, G., Alm, A., & Larsson, E. (2009). A population-based study of macular thickness in full-term children assessed with Stratus OCT: normative data and repeatability. Acta Ophthalmol, 87(7), 741-745.

Eriksson, U., Larsson, E., & Holmström, G. (2004). Optical coherence tomography in the diagnosis of juvenile X-linked retinoschisis. Acta Ophthalmol Scand, 82(2), 218-223.

Euler, T., Haverkamp, S., Schubert, T., & Baden, T. (2014). Retinal bipolar cells: elementary building blocks of vision. Nat Rev Neurosci, 15(8), 507-519.

EXPRESS Group. (2010). Incidence of and risk factors for neonatal morbidity after active perinatal care: extremely preterm infants study in Sweden (EXPRESS). Acta Paediatr, 99(7), 978-992.

71

Fellman V, H. W. L., Norman M, Westgren M, Källén K, Lagercrantz H, Marsal K, Serenius F, Wennergren M. (2009). One-year survival of extremely preterm infants after active perinatal care in Sweden. Jama, 301(21), 2225-2233.

Fledelius, H. C. (1996). Pre-term delivery and subsequent ocular development. A 7-10 year follow-up of children screened 1982-84 for ROP. 3) Refraction. Myopia of prematurity. Acta Ophthalmol Scand, 74(3), 297-300.

Fledelius, H. C., Bangsgaard, R., Slidsborg, C., & laCour, M. (2015). Refraction and visual acuity in a national Danish cohort of 4-year-old children of extremely preterm delivery. Acta Ophthalmol, 93(4), 330-338.

Flitcroft, D. I., Adams, G. G., Robson, A. G., & Holder, G. E. (2005). Retinal dysfunction and refractive errors: an electrophysiological study of children. Br J Ophthalmol, 89(4), 484-488.

Fujinami, K., Zernant, J., Chana, R. K., Wright, G. A., Tsunoda, K., Ozawa, Y., . . . Moore, A. T. (2015). Clinical and molecular characteristics of childhood-onset Stargardt disease. Ophthalmology, 122(2), 326-334.

Fulton, A. B., & Hansen, R. M. (1995). Electroretinogram responses and refractive errors in patients with a history of retinopathy prematurity. Doc Ophthalmol, 91(2), 87-100.

Fulton, A. B., Hansen, R. M., & Moskowitz, A. (2008). The cone electroretinogram in retinopathy of prematurity. Invest Ophthalmol Vis Sci, 49(2), 814-819.

Fulton, A. B., Hansen, R. M., Moskowitz, A., & Akula, J. D. (2009). The neurovascular retina in retinopathy of prematurity. Prog Retin Eye Res, 28(6), 452-482.

Fulton, A. B., Hansen, R. M., Moskowitz, A., & Barnaby, A. M. (2005). Multifocal ERG in subjects with a history of retinopathy of prematurity. Doc Ophthalmol, 111(1), 7-13.

Fulton, A. B., Hansen, R. M., Petersen, R. A., & Vanderveen, D. K. (2001). The rod photoreceptors in retinopathy of prematurity: an electroretinographic study. Arch Ophthalmol, 119(4), 499-505.

Gabriele, M. L., Wollstein, G., Ishikawa, H., Kagemann, L., Xu, J., Folio, L. S., & Schuman, J. S. (2011). Optical coherence tomography: history, current status, and laboratory work. Invest Ophthalmol Vis Sci, 52(5), 2425-2436.

Garcia-Martin, E., Pinilla, I., Idoipe, M., Fuertes, I., & Pueyo, V. (2011). Intra and interoperator reproducibility of retinal nerve fibre and macular thickness measurements using Cirrus Fourier-domain OCT. Acta Ophthalmol, 89(1), e23-29.

Garg, R., Agthe, A. G., Donohue, P. K., & Lehmann, C. U. (2003). Hyperglycemia and retinopathy of prematurity in very low birth weight infants. J Perinatol, 23(3), 186-194.

Gariano, R. F., Iruela-Arispe, M. L., & Hendrickson, A. E. (1994). Vascular development in primate retina: comparison of laminar plexus formation in monkey and human. Invest Ophthalmol Vis Sci, 35(9), 3442-3455.

Gerth, C., Sutter, E. E., & Werner, J. S. (2003). mfERG response dynamics of the aging retina. Invest Ophthalmol Vis Sci, 44(10), 4443-4450.

Gilbert, C. (2007). Changing challenges in the control of blindness in children. Eye (Lond), 21(10), 1338-1343.

Gränse, L., Ponjavic, V., & Andréasson, S. (2004). Full-field ERG, multifocal ERG and multifocal VEP in patients with retinitis pigmentosa and residual central visual fields. Acta Ophthalmol Scand, 82(6), 701-706.

72

Gultom, E., Doyle, L. W., Davis, P., Dharmalingam, A., & Bowman, E. (1997). Changes over time in attitudes to treatment and survival rates for extremely preterm infants (23-27 weeks' gestational age). Aust N Z J Obstet Gynaecol, 37(1), 56-58.

Hamilton, R., Bradnam, M. S., Dudgeon, J., & Mactier, H. (2008). Maturation of rod function in preterm infants with and without retinopathy of prematurity. J Pediatr, 153(5), 605-611.

Harris, M. E., Moskowitz, A., Fulton, A. B., & Hansen, R. M. (2011). Long-term effects of retinopathy of prematurity (ROP) on rod and rod-driven function. Doc Ophthalmol, 122(1), 19-27.

Haugen, O. H., Nepstad, L., Standal, O. A., Elgen, I., & Markestad, T. (2012). Visual function in 6 to 7 year-old children born extremely preterm: a population-based study. Acta Ophthalmol, 90(5), 422-427.

Haverkamp, S., Haeseleer, F., & Hendrickson, A. (2003). A comparison of immunocytochemical markers to identify bipolar cell types in human and monkey retina. Vis Neurosci, 20(6), 589-600.

Hedin, A., & Olsson, K. (1984). Letter legibility and the construction of a new visual acuity chart. Ophthalmologica, 189(3), 147-156.

Hee, M. R., Izatt, J. A., Swanson, E. A., Huang, D., Schuman, J. S., Lin, C. P., . . . Fujimoto, J. G. (1995). Optical coherence tomography of the human retina. Arch Ophthalmol, 113(3), 325-332.

Hellgren, K. M., Tornqvist, K., Jakobsson, P. G., Lundgren, P., Carlsson, B., Källén, K., . . . Holmström, G. (2016). Ophthalmologic Outcome of Extremely Preterm Infants at 6.5 Years of Age: Extremely Preterm Infants in Sweden Study (EXPRESS). JAMA Ophthalmol.

Hellström, A., Engström, E., Hård, A. L., Albertsson-Wikland, K., Carlsson, B., Niklasson, A., . . . Smith, L. E. (2003). Postnatal serum insulin-like growth factor I deficiency is associated with retinopathy of prematurity and other complications of premature birth. Pediatrics, 112(5), 1016-1020.

Hendrickson, A. (1992). A morphological comparison of foveal development in man and monkey. Eye (Lond), 6 ( Pt 2), 136-144.

Hendrickson, A. (2016). Development of Retinal Layers in Prenatal Human Retina. Am J Ophthalmol, 161, 29-35 e21.

Hendrickson, A., Bumsted-O'Brien, K., Natoli, R., Ramamurthy, V., Possin, D., & Provis, J. (2008). Rod photoreceptor differentiation in fetal and infant human retina. Exp Eye Res, 87(5), 415-426.

Hendrickson, A., & Drucker, D. (1992). The development of parafoveal and mid-peripheral human retina. Behav Brain Res, 49(1), 21-31.

Hendrickson, A., Possin, D., Vajzovic, L., & Toth, C. A. (2012). Histologic development of the human fovea from midgestation to maturity. Am J Ophthalmol, 154(5), 767-778 e762.

Hendrickson, A. E., & Yuodelis, C. (1984). The morphological development of the human fovea. Ophthalmology, 91(6), 603-612.

Hollenberg, M. J., & Spira, A. W. (1972). Early development of the human retina. Can J Ophthalmol, 7(4), 472-491.

Holm, K., Larsson, J., & Lövestam-Adrian, M. (2007). In diabetic retinopathy, foveal thickness of 300 mum seems to correlate with functionally significant loss of vision. Doc Ophthalmol, 114(3), 117-124.

Holmström, G., el Azazi, M., Jacobson, L., & Lennerstrand, G. (1993). A population based, prospective study of the development of ROP in prematurely born children in the Stockholm area of Sweden. Br J Ophthalmol, 77(7), 417-423.

73

Holmström, G., el Azazi, M., & Kugelberg, U. (1999). Ophthalmological follow up of preterm infants: a population based, prospective study of visual acuity and strabismus. Br J Ophthalmol, 83(2), 143-150.

Holmström, G., Eriksson, U., Hellgren, K., & Larsson, E. (2010). Optical coherence tomography is helpful in the diagnosis of foveal hypoplasia. Acta Ophthalmol, 88(4), 439-442.

Holmström, G., Hellström, A., Jakobsson, P., Lundgren, P., Tornqvist, K., & Wallin, A. (2015). Evaluation of new guidelines for ROP screening in Sweden using SWEDROP - a national quality register. Acta Ophthalmol, 93(3), 265-268.

Holmström, G., Hellström, A., Jakobsson, P., Lundgren, P., Tornqvist, K., & Wallin, A. (2016). Five years of treatment for retinopathy of prematurity in Sweden: results from SWEDROP, a national quality register. Br J Ophthalmol, 100(12), 1656-1661.

Holmström, G., & Larsson, E. (2008). Long-term follow-up of visual functions in prematurely born children--a prospective population-based study up to 10 years of age. J AAPOS, 12(2), 157-162.

Holmström, G. E., Källén, K., Hellström, A., Jakobsson, P. G., Serenius, F., Stjernqvist, K., & Tornqvist, K. (2014). Ophthalmologic outcome at 30 months' corrected age of a prospective Swedish cohort of children born before 27 weeks of gestation: the extremely preterm infants in sweden study. JAMA Ophthalmol, 132(2), 182-189.

Hood, D. C., Bach, M., Brigell, M., Keating, D., Kondo, M., Lyons, J. S., . . . Palmowski-Wolfe, A. M. (2012). ISCEV standard for clinical multifocal electroretinography (mfERG) (2011 edition). Doc Ophthalmol, 124(1), 1-13.

Hood, D. C., Frishman, L. J., Saszik, S., & Viswanathan, S. (2002). Retinal origins of the primate multifocal ERG: implications for the human response. Invest Ophthalmol Vis Sci, 43(5), 1673-1685.

Hood, D. C., Greenstein, V., Frishman, L., Holopigian, K., Viswanathan, S., Seiple, W., . . . Robson, J. G. (1999). Identifying inner retinal contributions to the human multifocal ERG. Vision Res, 39(13), 2285-2291.

Huang, D., Swanson, E. A., Lin, C. P., Schuman, J. S., Stinson, W. G., Chang, W., . . . et al. (1991). Optical coherence tomography. Science, 254(5035), 1178-1181.

Hughes, S., Yang, H., & Chan-Ling, T. (2000). Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis. Invest Ophthalmol Vis Sci, 41(5), 1217-1228.

Husain, S. M., Sinha, A. K., Bunce, C., Arora, P., Lopez, W., Mun, K. S., . . . Adams, G. G. (2013). Relationships between maternal ethnicity, gestational age, birth weight, weight gain, and severe retinopathy of prematurity. J Pediatr, 163(1), 67-72.

Hyvärinen, L., Näsänen, R., & Laurinen, P. (1980). New visual acuity test for pre-school children. Acta Ophthalmol (Copenh), 58(4), 507-511.

Håkansson, S., Farooqi, A., Holmgren, P. A., Serenius, F., & Högberg, U. (2004). Proactive management promotes outcome in extremely preterm infants: a population-based comparison of two perinatal management strategies. Pediatrics, 114(1), 58-64.

Jackman, S. L., Babai, N., Chambers, J. J., Thoreson, W. B., & Kramer, R. H. (2011). A positive feedback synapse from retinal horizontal cells to cone photoreceptors. PLoS Biol, 9(5), e1001057.

Jackson, G. R., Ortega, J., Girkin, C., Rosenstiel, C. E., & Owsley, C. (2002). Aging-related changes in the multifocal electroretinogram. J Opt Soc Am A Opt Image Sci Vis, 19(1), 185-189.

74

Jackson, R. A., Gibson, K. A., Wu, Y. W., & Croughan, M. S. (2004). Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol, 103(3), 551-563.

Jacobs, G. H. (1996). Primate photopigments and primate color vision. Proc Natl Acad Sci U S A, 93(2), 577-581.

Jacobson, L., Ygge, J., & Flodmark, O. (1998). Nystagmus in periventricular leucomalacia. Br J Ophthalmol, 82(9), 1026-1032.

Jacobson, L. K., & Dutton, G. N. (2000). Periventricular leukomalacia: an important cause of visual and ocular motility dysfunction in children. Surv Ophthalmol, 45(1), 1-13.

James, S., & Lanman, J. T. (1976). History of oxygen therapy and retrolental fibroplasia. Prepared by the American Academy of Pediatrics, Committee on Fetus and Newborn with the collaboration of special consultants. Pediatrics, 57(suppl 2), 591-642.

Kamper, J., Feilberg Jorgensen, N., Jonsbo, F., Pedersen-Bjergaard, L., Pryds, O., & Danish, E. S. G. (2004). The Danish national study in infants with extremely low gestational age and birthweight (the ETFOL study): respiratory morbidity and outcome. Acta Paediatr, 93(2), 225-232.

Kefalov, V., Fu, Y., Marsh-Armstrong, N., & Yau, K. W. (2003). Role of visual pigment properties in rod and cone phototransduction. Nature, 425(6957), 526-531.

Kjellström, S., Vijayasarathy, C., Ponjavic, V., Sieving, P. A., & Andreasson, S. (2010). Long-term 12 year follow-up of X-linked congenital retinoschisis. Ophthalmic Genet, 31(3), 114-125.

Klebanoff, M. A., & Keim, S. A. (2011). Epidemiology: the changing face of preterm birth. Clin Perinatol, 38(3), 339-350.

Kolb, H. (1995). Midget pathways of the primate retina underlie resolution and red green color opponency. In H. Kolb, E. Fernandez, & R. Nelson (Eds.), Webvision: The Organization of the Retina and Visual System. Salt Lake City (UT).

Kolb, H., & Marshak, D. (2003). The midget pathways of the primate retina. Doc Ophthalmol, 106(1), 67-81.

Koustenis, A., Jr., Harris, A., Gross, J., Januleviciene, I., Shah, A., & Siesky, B. (2017). Optical coherence tomography angiography: an overview of the technology and an assessment of applications for clinical research. Br J Ophthalmol, 101(1), 16-20.

Kozulin, P., Natoli, R., Bumsted O'Brien, K. M., Madigan, M. C., & Provis, J. M. (2010). The cellular expression of antiangiogenic factors in fetal primate macula. Invest Ophthalmol Vis Sci, 51(8), 4298-4306.

Kraft, T. W., Schneeweis, D. M., & Schnapf, J. L. (1993). Visual transduction in human rod photoreceptors. J Physiol, 464, 747-765.

Lagercrantz, H., Hellström-Westas, L., & Norman, M. (2015). Neonatologi. Studentlitteratur AB, Lund.

Larsson, E., & Holmström, G. (2002). Screening for retinopathy of prematurity: evaluation and modification of guidelines. Br J Ophthalmol, 86(12), 1399-1402.

Larsson, E. K., Rydberg, A. C., & Holmström, G. E. (2003). A population-based study of the refractive outcome in 10-year-old preterm and full-term children. Arch Ophthalmol, 121(10), 1430-1436.

Lee, K. M., Kim, J. H., & Yu, Y. S. (2010). Idiopathic maculopathy in eyes with regressed retinopathy of prematurity. Graefes Arch Clin Exp Ophthalmol, 248(8), 1097-1103.

75

Lennartsson, F., Nilsson, M., Flodmark, O., & Jacobson, L. (2014). Damage to the immature optic radiation causes severe reduction of the retinal nerve fiber layer, resulting in predictable visual field defects. Invest Ophthalmol Vis Sci, 55(12), 8278-8288.

Linberg, K. A., & Fisher, S. K. (1990). A burst of differentiation in the outer posterior retina of the eleven-week human fetus: an ultrastructural study. Vis Neurosci, 5(1), 43-60.

Lineham, J. D., Smith, R. M., Dahlenburg, G. W., King, R. A., Haslam, R. R., Stuart, M. C., & Faull, L. (1986). Circulating insulin-like growth factor I levels in newborn premature and full-term infants followed longitudinally. Early Hum Dev, 13(1), 37-46.

Liu, G., Li, H., Liu, X., Xu, D., & Wang, F. (2016). Structural analysis of retinal photoreceptor ellipsoid zone and postreceptor retinal layer associated with visual acuity in patients with retinitis pigmentosa by ganglion cell analysis combined with OCT imaging. Medicine (Baltimore), 95(52), e5785.

Maldonado, R. S., O'Connell, R. V., Sarin, N., Freedman, S. F., Wallace, D. K., Cotten, C. M., . . . Toth, C. A. (2011). Dynamics of human foveal development after premature birth. Ophthalmology, 118(12), 2315-2325.

Mann, I. (1964). The development of the human eye (3d ed.). New York,: Grune & Stratton.

Mansson, J., & Stjernqvist, K. (2014). Children born extremely preterm show significant lower cognitive, language and motor function levels compared with children born at term, as measured by the Bayley-III at 2.5 years. Acta Paediatr, 103(5), 504-511.

Markestad, T., Kaaresen, P. I., Ronnestad, A., Reigstad, H., Lossius, K., Medbo, S., . . . Norwegian Extreme Prematurity Study, G. (2005). Early death, morbidity, and need of treatment among extremely premature infants. Pediatrics, 115(5), 1289-1298.

Marmor, M. F., Choi, S. S., Zawadzki, R. J., & Werner, J. S. (2008). Visual insignificance of the foveal pit: reassessment of foveal hypoplasia as fovea plana. Arch Ophthalmol, 126(7), 907-913.

McCulloch, D. L., Marmor, M. F., Brigell, M. G., Hamilton, R., Holder, G. E., Tzekov, R., & Bach, M. (2015). ISCEV Standard for full-field clinical electroretinography (2015 update). Doc Ophthalmol, 130(1), 1-12.

McLoone, E., O'Keefe, M., McLoone, S., & Lanigan, B. (2006). Long term functional and structural outcomes of laser therapy for retinopathy of prematurity. Br J Ophthalmol, 90(6), 754-759.

Mercer, B. M., Goldenberg, R. L., Moawad, A. H., Meis, P. J., Iams, J. D., Das, A. F., . . . McNellis, D. (1999). The preterm prediction study: effect of gestational age and cause of preterm birth on subsequent obstetric outcome. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Am J Obstet Gynecol, 181(5 Pt 1), 1216-1221.

Michaelides, M., Hunt, D. M., & Moore, A. T. (2004). The cone dysfunction syndromes. Br J Ophthalmol, 88(2), 291-297.

Michalczuk, M., Urban, B., Chrzanowska-Grenda, B., Ozieblo-Kupczyk, M., Bakunowicz-Lazarczyk, A., & Kretowska, M. (2016). The assessment of multifocal ERG responses in school-age children with history of prematurity. Doc Ophthalmol, 132(1), 47-55.

Mintz-Hittner, H. A., Kennedy, K. A., Chuang, A. Z., & Group, B.-R. C. (2011). Efficacy of intravitreal bevacizumab for stage 3+ retinopathy of prematurity. N Engl J Med, 364(7), 603-615.

76

Mintz-Hittner, H. A., Knight-Nanan, D. M., Satriano, D. R., & Kretzer, F. L. (1999). A small foveal avascular zone may be an historic mark of prematurity. Ophthalmology, 106(7), 1409-1413.

Munaut, C., Lambert, V., Noel, A., Frankenne, F., Deprez, M., Foidart, J. M., & Rakic, J. M. (2001). Presence of oestrogen receptor type beta in human retina. Br J Ophthalmol, 85(7), 877-882.

Mylonas, G., Ahlers, C., Malamos, P., Golbaz, I., Deak, G., Schuetze, C., . . . Schmidt-Erfurth, U. (2009). Comparison of retinal thickness measurements and segmentation performance of four different spectral and time domain OCT devices in neovascular age-related macular degeneration. Br J Ophthalmol, 93(11), 1453-1460.

O'Connor, A. R., Stephenson, T., Johnson, A., Tobin, M. J., Moseley, M. J., Ratib, S., . . . Fielder, A. R. (2002). Long-term ophthalmic outcome of low birth weight children with and without retinopathy of prematurity. Pediatrics, 109(1), 12-18.

O'Rahilly, R. (1975). The prenatal development of the human eye. Exp Eye Res, 21(2), 93-112.

Ospina, L. H., Lyons, C. J., Matsuba, C., Jan, J., & McCormick, A. Q. (2005). Argon laser photocoagulation for retinopathy of prematurity: long-term outcome. Eye (Lond), 19(11), 1213-1218.

Ozawa, G. Y., Bearse, M. A., Jr., Harrison, W. W., Bronson-Castain, K. W., Schneck, M. E., Barez, S., & Adams, A. J. (2014). Differences in neuroretinal function between adult males and females. Optom Vis Sci, 91(6), 602-607.

Papile, L. A., Burstein, J., Burstein, R., & Koffler, H. (1978). Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weights less than 1,500 gm. J Pediatr, 92(4), 529-534.

Patz, A., Hoeck, L. E., & De La Cruz, E. (1952). Studies on the effect of high oxygen administration in retrolental fibroplasia. I. Nursery observations. Am J Ophthalmol, 35(9), 1248-1253.

Provis, J. M. (2001). Development of the primate retinal vasculature. Prog Retin Eye Res, 20(6), 799-821.

Provis, J. M., & Hendrickson, A. E. (2008). The foveal avascular region of developing human retina. Arch Ophthalmol, 126(4), 507-511.

Provis, J. M., Leech, J., Diaz, C. M., Penfold, P. L., Stone, J., & Keshet, E. (1997). Development of the human retinal vasculature: cellular relations and VEGF expression. Exp Eye Res, 65(4), 555-568.

Provis, J. M., Sandercoe, T., & Hendrickson, A. E. (2000). Astrocytes and blood vessels define the foveal rim during primate retinal development. Invest Ophthalmol Vis Sci, 41(10), 2827-2836.

Quinn, G. E., Dobson, V., Davitt, B. V., Wallace, D. K., Hardy, R. J., Tung, B., . . . Early Treatment for Retinopathy of Prematurity Cooperative, G. (2013). Progression of myopia and high myopia in the Early Treatment for Retinopathy of Prematurity study: findings at 4 to 6 years of age. J AAPOS, 17(2), 124-128.

Raffa, L. H., Nilsson, J., Dahlgren, J., & Grönlund, M. A. (2017). Electrophysiological changes in 12-year-old children born MLP: reduced VEP amplitude in MLP children. Br J Ophthalmol.

Rapp, L. M., Maple, S. S., & Choi, J. H. (2000). Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina. Invest Ophthalmol Vis Sci, 41(5), 1200-1209.

Reichenbach, A., & Bringmann, A. (2013). New functions of Muller cells. Glia, 61(5), 651-678.

77

Robson, J. G., & Frishman, L. J. (1998). Dissecting the dark-adapted electroretinogram. Doc Ophthalmol, 95(3-4), 187-215.

Rosén, R., Sjöstrand, J., Nilsson, M., & Hellgren, K. (2015). A methodological approach for evaluation of foveal immaturity after extremely preterm birth. Ophthalmic Physiol Opt, 35(4), 433-441.

Schnapf, J. L., Kraft, T. W., & Baylor, D. A. (1987). Spectral sensitivity of human cone photoreceptors. Nature, 325(6103), 439-441.

Seiple, W., Vajaranant, T. S., Szlyk, J. P., Clemens, C., Holopigian, K., Paliga, J., . . . Carr, R. E. (2003). Multifocal electroretinography as a function of age: the importance of normative values for older adults. Invest Ophthalmol Vis Sci, 44(4), 1783-1792.

Serenius, F., Ewald, U., Farooqi, A., Fellman, V., Hafstrom, M., Hellgren, K., . . . Extremely Preterm Infants in Sweden Study, G. (2016). Neurodevelopmental Outcomes Among Extremely Preterm Infants 6.5 Years After Active Perinatal Care in Sweden. JAMA Pediatr, 170(10), 954-963.

Serenius, F., Källén, K., Blennow, M., Ewald, U., Fellman, V., Holmström, G., . . . Strömberg, B. (2013). Neurodevelopmental outcome in extremely preterm infants at 2.5 years after active perinatal care in Sweden. Jama, 309(17), 1810-1820.

Smith, L. E., Wesolowski, E., McLellan, A., Kostyk, S. K., D'Amato, R., Sullivan, R., & D'Amore, P. A. (1994). Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci, 35(1), 101-111.

Song, A. P., Wu, X. Y., Wang, J. R., Liu, W., Sun, Y., & Yu, T. (2014). Measurement of retinal thickness in macular region of high myopic eyes using spectral domain OCT. Int J Ophthalmol, 7(1), 122-127.

Song, W. K., Lee, S. C., Lee, E. S., Kim, C. Y., & Kim, S. S. (2010). Macular thickness variations with sex, age, and axial length in healthy subjects: a spectral domain-optical coherence tomography study. Invest Ophthalmol Vis Sci, 51(8), 3913-3918.

Soong, G. P., Shapiro, M., Seiple, W., & Szlyk, J. P. (2008). Macular structure and vision of patients with macular heterotopia secondary to retinopathy of prematurity. Retina, 28(8), 1111-1116.

Staurenghi, G., Sadda, S., Chakravarthy, U., Spaide, R. F., & International Nomenclature for Optical Coherence Tomography, P. (2014). Proposed lexicon for anatomic landmarks in normal posterior segment spectral-domain optical coherence tomography: the IN*OCT consensus. Ophthalmology, 121(8), 1572-1578.

Steinberg, R. H. (1985). Interactions between the retinal pigment epithelium and the neural retina. Doc Ophthalmol, 60(4), 327-346.

Stockton, R. A., & Slaughter, M. M. (1989). B-wave of the electroretinogram. A reflection of ON bipolar cell activity. J Gen Physiol, 93(1), 101-122.

Sutter, E. E., & Tran, D. (1992). The field topography of ERG components in man--I. The photopic luminance response. Vision Res, 32(3), 433-446.

Sutton, L., & Bajuk, B. (1999). Population-based study of infants born at less than 28 weeks' gestation in New South Wales, Australia, in 1992-3. New South Wales Neonatal Intensive Care Unit Study Group. Paediatr Perinat Epidemiol, 13(3), 288-301.

Swanson, E. A., Izatt, J. A., Hee, M. R., Huang, D., Lin, C. P., Schuman, J. S., . . . Fujimoto, J. G. (1993). In vivo retinal imaging by optical coherence tomography. Opt Lett, 18(21), 1864-1866.

78

Tariq, Y. M., Burlutsky, G., & Mitchell, P. (2012). Macular parameters and prematurity: A spectral domain coherence tomography study. J AAPOS, 16(4), 382-385.

Terry, T. L. (1942). Fibroblastic Overgrowth of Persistent Tunica Vasculosa Lentis in Infants Born Prematurely: II. Report of Cases-Clinical Aspects. Trans Am Ophthalmol Soc, 40, 262-284.

Testa, F., Maguire, A. M., Rossi, S., Pierce, E. A., Melillo, P., Marshall, K., . . . Simonelli, F. (2013). Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2. Ophthalmology, 120(6), 1283-1291.

Thomas, M. G., Kumar, A., Mohammad, S., Proudlock, F. A., Engle, E. C., Andrews, C., . . . Gottlob, I. (2011). Structural grading of foveal hypoplasia using spectral-domain optical coherence tomography a predictor of visual acuity? Ophthalmology, 118(8), 1653-1660.

Tolsma, K. W., Allred, E. N., Chen, M. L., Duker, J., Leviton, A., & Dammann, O. (2011). Neonatal bacteremia and retinopathy of prematurity: the ELGAN study. Arch Ophthalmol, 129(12), 1555-1563.

Toth, C. A., Narayan, D. G., Boppart, S. A., Hee, M. R., Fujimoto, J. G., Birngruber, R., . . . Roach, W. P. (1997). A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy. Arch Ophthalmol, 115(11), 1425-1428.

Tunon, K., Eik-Nes, S. H., & Grottum, P. (1996). A comparison between ultrasound and a reliable last menstrual period as predictors of the day of delivery in 15,000 examinations. Ultrasound Obstet Gynecol, 8(3), 178-185.

Vajzovic, L., Hendrickson, A. E., O'Connell, R. V., Clark, L. A., Tran-Viet, D., Possin, D., . . . Toth, C. A. (2012). Maturation of the human fovea: correlation of spectral-domain optical coherence tomography findings with histology. Am J Ophthalmol, 154(5), 779-789 e772.

Vajzovic, L., Rothman, A. L., Tran-Viet, D., Cabrera, M. T., Freedman, S. F., & Toth, C. A. (2015). Delay in retinal photoreceptor development in very preterm compared to term infants. Invest Ophthalmol Vis Sci, 56(2), 908-913.

Vanhaesebrouck, P., Allegaert, K., Bottu, J., Debauche, C., Devlieger, H., Docx, M., . . . Extremely Preterm Infants in Belgium Study, G. (2004). The EPIBEL study: outcomes to discharge from hospital for extremely preterm infants in Belgium. Pediatrics, 114(3), 663-675.

Verdon, W. A., & Haegerstrom-Portnoy, G. (1998). Topography of the multifocal electroretinogram. Doc Ophthalmol, 95(1), 73-90.

von Hanno, T., Lade, A. C., Mathiesen, E. B., Peto, T., Njolstad, I., & Bertelsen, G. (2016). Macular thickness in healthy eyes of adults (N = 4508) and relation to sex, age and refraction: the Tromso Eye Study (2007-2008). Acta Ophthalmol.

Walsh, B. K., Brooks, T. M., & Grenier, B. M. (2009). Oxygen therapy in the neonatal care environment. Respir Care, 54(9), 1193-1202.

Wang, J., Spencer, R., Leffler, J. N., & Birch, E. E. (2012). Critical period for foveal fine structure in children with regressed retinopathy of prematurity. Retina, 32(2), 330-339.

Wojtkowski, M., Leitgeb, R., Kowalczyk, A., Bajraszewski, T., & Fercher, A. F. (2002). In vivo human retinal imaging by Fourier domain optical coherence tomography. J Biomed Opt, 7(3), 457-463.

Wood, N. S., Marlow, N., Costeloe, K., Gibson, A. T., & Wilkinson, A. R. (2000). Neurologic and developmental disability after extremely preterm birth. EPICure Study Group. N Engl J Med, 343(6), 378-384.

79

Wu, W. C., Lin, R. I., Shih, C. P., Wang, N. K., Chen, Y. P., Chao, A. N., . . . Tsai, S. (2012). Visual acuity, optical components, and macular abnormalities in patients with a history of retinopathy of prematurity. Ophthalmology, 119(9), 1907-1916.

Yanni, S. E., Wang, J., Chan, M., Carroll, J., Farsiu, S., Leffler, J. N., . . . Birch, E. E. (2012). Foveal avascular zone and foveal pit formation after preterm birth. Br J Ophthalmol, 96(7), 961-966.

Yau, K. W. (1994). Phototransduction mechanism in retinal rods and cones. The Friedenwald Lecture. Invest Ophthalmol Vis Sci, 35(1), 9-32.

Yokouchi, H., Baba, T., Misawa, S., Kitahashi, M., Oshitari, T., Kuwabara, S., & Yamamoto, S. (2016). Changes in subfoveal choroidal thickness and reduction of serum levels of vascular endothelial growth factor in patients with POEMS syndrome. Br J Ophthalmol.

Young, R. W. (1985). Cell differentiation in the retina of the mouse. Anat Rec, 212(2), 199-205.

Yu, D. Y., Cringle, S. J., Balaratnasingam, C., Morgan, W. H., Yu, P. K., & Su, E. N. (2013). Retinal ganglion cells: Energetics, compartmentation, axonal transport, cytoskeletons and vulnerability. Prog Retin Eye Res, 36, 217-246.

Yuodelis, C., & Hendrickson, A. (1986). A qualitative and quantitative analysis of the human fovea during development. Vision Res, 26(6), 847-855.

Zhou, X., Huang, X., Chen, H., & Zhao, P. (2010). Comparison of electroretinogram between healthy preterm and term infants. Doc Ophthalmol, 121(3), 205-213.

Åkerblom, H., Andréasson, S., & Holmstrom, G. (2016). Macular function in preterm children at school age. Doc Ophthalmol, 133(3), 151-157.

Åkerblom, H., Andréasson, S., Larsson, E., & Holmström, G. (2014). Photoreceptor Function in School-Aged Children is Affected by Preterm Birth. Transl Vis Sci Technol, 3(6), 7.

Åkerblom, H., Holmström, G., Eriksson, U., & Larsson, E. (2012). Retinal nerve fibre layer thickness in school-aged prematurely-born children compared to children born at term. Br J Ophthalmol, 96(7), 956-960.

Åkerblom, H., Larsson, E., Eriksson, U., & Holmström, G. (2011). Central macular thickness is correlated with gestational age at birth in prematurely born children. Br J Ophthalmol, 95(6), 799-803.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1318

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-317550

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017