Novel Opsins and Oxygen Induced Retinopathy Open Access

Abstract

Background: Retinopathy of prematurity (ROP) is a major complication of preterm birth that can lead to different degrees of vision impairment. The ROP spectrum ranges from mild forms that may resolve spontaneously or advanced stage diagnoses indicative of retinal detachment and subsequent blindness. Evidence suggests that light activation of OPN4 and OPN5 may be used to reduce neovascularization in a mouse model of ROP. This study serves as a launching point for an investigation of novel opsins and OIR.

Methods: Studying oxygen-induced retinopathy (OIR) in mice has been a valuable mode of investigating the pathology of ROP and treatment options in a pre-clinical in vivo model. Retinopathy was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12 in wild type C57BL/6J mice and Opn4 and Opn5 mutant mice. Wild type C57BL/6J mice were sacrificed at P17 and the Opn4 line was sacrificed at P12. To quantify vasculature, a novel technique for 3D analysis was developed using Imaris software. 

Results: Retinal vasculature of OIR mice occupied a smaller area, were less volumetric, and exhibited fewer branch points than control mice at P17. Wild type C57BL/6J mice did not show quantifiable evidence of neovascularization in the periphery of the retina. Preliminary images from the P12 Opn4 line serve as visual evidence of an avascular zone at the center of the retina.

Conclusion: The results suggest that some degree of retinopathy was induced in wild type C57BL/6J mice and the Opn4 line. Continued experiments on wild type and genetically modified mice that quantify OIR at different points of retinal development is imperative before applying light therapy as a treatment option.

Table of Contents

Abstract…………………………………..………………………………………………………………………………………………..1

1. Introduction..…………………………………………………………………………………………………………………………2

           1.1 Background and Overview of ROP…………………………………………………………………………….2

1.2 Pathology of ROP………………………………………………….………………………………………………….3

1.3 Oxygen-Induced Retinopathy (OIR) as a Model.……….…………..…………………..……………..4

1.4 Quantification of Vasculature……….…………………………………………..……………………………..5

1.5 Oxygen Therapy..………………………………………………….………………………………………………….6

1.6 Anti-Angiogenesis Therapy..………………………………….………………………………………………….7

1.7 Melanopsin and Light………..………………………………….………………………………………………….8

1.8 Neuropsin and Light………..……..…………………………….………………………………………………..10

2. Objective and hypothesis ………………………………………………………………………………………………….…11

3. Methods …………………………………………………………………………………………………………………………..…12

3.1 Study Approval and Subjects……..…………………………………………………………………………..12

3.2 OIR Protocol……..……………………………………………………………………………………………..…….13

3.3 Whole Retina Flatmounting……..…………………………………………………………………………….14

3.4 Microscopy……..……………………..………………………………………………………………………..…….15

3.5 Quantification of Vasculature…………………………………………………………………………..…….15

3.6 Statistical Analysis……..……………..……………………………………………………………………..…….16

4. Results …………………………………………………………………………………………………………………………..……17

4.1 Visual Comparison Between C57BL/6J WT Room Air (RA) vs. OIR Images at P17……17

Figure 1A. RA vs. OIR C57BL/6J WT retina flatmounts……………………….……………..18

Figure 1B. RA vs. OIR C57BL/6J WT retina flatmounts………………………………….…..19

4.2 Morphometric Quantification of C57BL/6J WT Retinas with Imaris………………………..20

Figure 2. Analysis of flatmounts using Imaris software……………………………………..21

Figure 3. Vascular area in central and peripheral retina in RA vs. OIR Mice.……..22

Figure 4. Vascular volume in central and peripheral retina in RA vs. OIR Mice….23

Figure 5. Number of branch points in central and peripheral retina in RA vs. OIR Mice………………………………………………………………………………………………………………….24

4.3 Quantification of C57BL/6J WT Retinas with ImageJ……………………….…………………..….25

Figure 6. Whole retina CTCF in RA vs. OIR Mice ………………………………….…………...26 

4.4 Visual Comparison Between Opn4 wild type(WT; Opn4^+/+) and heterozygous (HT; Opn4 ^-/+) mice: RA vs. OIR Images at P12…………………….………………………………………………..27

           Figure 7. RA and OIR Opn4 WT/HT retina flatmounts……………….…………………………………28

5. Discussion.……………….……………………………………………………………………………………………………..…..29

5.1 Limitations………..……………….……………………………………………………………………………..……31

           5.2 Future Directions……………….……………………………………………………………………………..……32

6. References.. ……………….…………………………………………………………………………………………………..…..35

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience Biology, Physiology
Keyword	Retinopathy Ophthalmology Eye
Committee Chair / Thesis Advisor	Michael Iuvone , Emory University
Committee Members	Daniel Dilks, Emory University LaTonia Taliaferro-Smith, Emory University Donald Stein , Emory University

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