- Title
- Influence of lens induced myopia on retinal gene expression in the guinea pig
- Creator
- Fuchs, Dominique Lena
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Myopia is a refractive error characterized by an abnormal increase in eye length. The prevalence of myopia is increasing in a global manner and the increase in humans affected by myopia comes along with an increasing number of patients affected with high myopia (more than -6D). High myopia is associated with serious complications including retinal detachment, glaucoma and posterior staphyloma. Ocular development depends on visual experience. Myopia can be induced in animal eyes by rearing them with negative spectacle lenses which displaces the focal point behind the retina. The eye compensates for this imposed defocus by elongating more rapidly. Such visual manipulations enable the exploration of the so far poorly understood underlying biological mechanisms of myopia development. Myopia development is associated with transcription changes and physical changes in ocular tissues. This thesis focusses on the exploration of mRNA expression changes in the guinea pig retina that accompany changes in axial elongation. Therefore, myopic and non-myopic guinea pig retinas were compared via the high throughput method of RNA sequencing (RNA-seq). RNA-seq was used to compare normal retinal samples from untreated age-matched guinea pigs with those from guinea pigs that developed myopia over 4 or 11 days, and with samples originating from eyes that had recovered for 48 hours from the induced myopia. The RNA-seq results were analysed with 4 different downstream analysis tools (Voom, DESeq2, EdgeR and Cuffdiff) to determine differentially expressed genes (DEG’s). The results from the tools Cuffdiff were used for pathway analysis. Expression analysis with Cuffdiff resulted in 137 DEG’s after 4 days of myopia development, 98 DEG’s after 11 days of myopia development and 47 DEG’s after 48h of recovery from myopia development. Retinol binding protein 1 was significantly upregulated according to the corrected p-value in all 3 comparisons. Several of the DEG’s (FAM107A, Col4A4, ApoE, MMP-2, and RBP1) were successfully validated using quantitative real time polymerase chain reaction (qPCR). Some of the DEG’s after induced myopia which were determined by Cuffdiff are well known to play a role in scleral remodelling during myopia development: Bone morphogenetic protein 2, Metalloproteinase 1 (MMP-2) and collagens. MMP-2 was significantly upregulated after 11 days of myopia development, BMP-2 after 4 days of myopia development and a number of collagens were upregulated in both conditions. Pathway analysis revealed that the pathways RAR activation and Retinol biosynthesis were among the top 10 associated pathways after 4 days of myopia development and after 48h of recovery from myopia development. Another pathway related to Vitamin A metabolism, Retinoate biosynthesis, was among the top 10 associated pathways after 4 days of myopia development. Other significant top associated pathways after 4 days of induced myopia were related to eukaryotic translation initiation and regulation (EIF2 Signaling, Regulation of eIF4 and p70S6K Signaling 2, mTor signaling). Vitamin A related pathways were also activated after 48 hrs of recovery. Additional novel pathways among the top 10 associated pathways after 48h of recovery from myopia included gap junction signaling and Il-8 signaling. The 3 most associated pathways after 11 days of myopia development were Hepatic fibrosis, GP6 signaling Pathway, and intrinsic Prothrombin Activation. A closer look at the participating genes from the dataset in these 3 pathways revealed that the majority were collagens and in the case of GP6 signaling, exclusively a set of Collagens. Apolipoprotein D (ApoD), Collagen 4A4 (Col4A4), Cellular Retinol Binding Protein 1 (CRABP1) and Retinol Binding Protein 1 (RBP1) were located in the guinea pig retina via immunohistochemistry (IHC), and the presence of the corresponding proteins in the retina were shown for all. mRNA for both ApoD and RBP1 were also located in the retina using in situ hybridization (ISH). Müller cells were positive for CRABP1 and RBP1. Col4A4 and ApoD clearly stained cell bodies in the ganglion cell layer of the retina. The results of this study underpin previous findings that Retinoic acid (RA) signalling is involved in axial elongation. The findings also suggest that Müller cells might be involved in RA mediated processes due to axial elongation. Furthermore, the results of this study suggest that there might be a set of identical gene products that mediate defocus signals from the retina to the sclera. Moreover, the RNA-seq results demonstrate a large and diverse range of genes are involved in eye growth, with a large pool of downstream analysis possibilities. These possibilities can be the base of future studies that further explore the retinal transcriptome in conditions of elongated and inhibited eye growth.
- Subject
- myopia; gene expression; pathway analysis; retinol biosynthesis
- Identifier
- http://hdl.handle.net/1959.13/1408615
- Identifier
- uon:35863
- Rights
- Copyright 2019 Dominique Lena Fuchs
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 8 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 1 MB | Adobe Acrobat PDF | View Details Download |