Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/ choroid in chick model of form-deprivation myopia

Purpose RNA sequencing analysis has demonstrated bidirectional changes in metabolism, structural and immune pathways during early induction of defocus induced myopia. Thus, the aim of this study was to investigate whether similar gene pathways are also related to the more excessive axial growth, ultrastructural and elemental microanalytic changes seen during the induction and recovery from form-deprivation myopia (FDM) in chicks and predicted by the RIDE model of myopia. Methods Archived genomic transcriptome data from the first three days of induction of monocularly occluded form deprived myopia (FDMI) in chicks was obtained from the GEO database (accession # GSE6543) while data from chicks monocularly occluded for 10 days and then given up to 24 h of normal visual recovery (FDMR) were collected. Gene set enrichment analysis (GSEA) software was used to determine enriched pathways during the induction (FDMI) and recovery (FDMR) from FD. Curated gene-sets were obtained from open access sources. Results Clusters of significant changes in mitochondrial energy metabolism, neurotransmission, ion channel transport, G protein coupled receptor signalling, complement cascades and neuron structure and growth were identified during the 10 days of induction of profound myopia and were found to correlate well with change in axial dimensions. Bile acid and bile salt metabolism pathways (cholesterol/lipid metabolism and sodium channel activation) were significantly upregulated during the first 24 h of recovery from 10 days of FDM. Conclusions The gene pathways altered during induction of FDM are similar to those reported in defocus induced myopia and are established indicators of oxidative stress, osmoregulatory and associated structural changes. These findings are also consistent with the choroidal thinning, axial elongation and hyperosmotic ion distribution patterns across the retina and choroid previously reported in FDM and predicted by RIDE

Retinal Mechanisms Controlling Eye Growth in Experimental Myopia

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Psychology and Public Health, College of Science, Health and Engineering, La Trobe University, Victoria, Australia

Bidirectional Expression of Metabolic, Structural, and Immune Pathways in Early Myopia and Hyperopia

Myopia (short-sightedness) affects 1.45 billion people worldwide, many of whom will develop sight-threatening secondary disorders. Myopic eyes are characterized by excessive size while hyperopic (long-sighted) eyes are typically small. The biological and genetic mechanisms underpinning the retina’s local control of these growth patterns remain unclear. In the present study, we used RNA sequencing to examine gene expression in the retina/RPE/choroid across 3 days of optically-induced myopia and hyperopia induction in chick. Data were analysed for differential expression of single genes, and Gene Set Enrichment Analysis (GSEA) was used to identify gene sets correlated with ocular axial length and refraction across lens groups. Like previous studies, we found few single genes that were differentially-expressed in a sign-of-defocus dependent manner (only BMP2 at 1 day). Using GSEA, however, we are the first to show that more subtle shifts in structural, metabolic, and immune pathway expression are correlated with the eye size and refractive changes induced by lens defocus. Our findings link gene expression with the morphological characteristics of refractive error, and suggest that physiological stress arising from metabolic and inflammatory pathway activation could increase the vulnerability of myopic eyes to secondary pathologie

Anti-diuretic hormone in the regulation of ocular volume in compensation to defocus

Abstract Purpose: The hormone Arginine Vasopressin (AVP) is a vasoconstrictor and anti-diuretic that is commonly associated with stress. Our previous results show that AVP causes a myopic shift in refractive compensation (RC) to +10D defocus (ARVO, 2013). Further, environmental stress in the form of asymmetric flicker impacts ocular growth (Crewther et al, 2006). Thus the current experiment aimed to investigate whether AVP plays a role in RC to defocus, and whether flicker affects this process. Methods: Experiment 1: RNA was extracted from the retina/RPE/choroid of chicks with + or -10D, or no defocus on days 5-7 post-hatching (n = 3 per lens group, per day) and prepared for sequencing on the Illumina HiSeq1500. Raw reads were mapped onto the chick genome and counts determined for each gene. Counts per million were imported into Pathway studio and GSEA conducted using the Mann-Whitney U-test algorithm (p<.05).<br /> Experiment 2: Chicks (n=360) were raised from day 5-9, with or without asymmetric flicker in the 12 hr day cycle, with + or -10D defocus (or non lens), following intravitreal injection of 5µl of either PBS, AVP or the AVP receptor antagonist ([des-Gly9-β-Mercapto-β, β cyclopentamethylenepropionyl1, O-Et-Tyr2,Val4,Arg8]-Vasopressin) (in PBS) into the experimental eye. Fellow eyes were injected with PBS. Retinoscopy and A-scan ultrasonography was performed on day 9. Tissue was collected and prepared for immunohistochemistry to examine AQP-4 and Kir4.1 expression. Results: Experiment 1: RNAseq revealed sign-dependent changes in AVP-related pathways over 3 days of rearing with defocus.<br /> Experiment 2: Flicker alone induced a myopic shift in both lens conditions. Flicker+AVP reduced hyperopia, axial elongation and anterior chamber depth in +10D lenses. Flicker+AVP antagonist reduced RC and ocular growth to -10D lenses. Immunohistochemistry showed altered AQP-4 and Kir4.1 staining across flicker conditions. Conclusions: Results indicate that changes in AVP-related gene expression occur concomitantly with changes in ocular volume during the induction of RC. Further, AVP and its antagonist also differentially interfered with the typical pattern of compensation to lenswear. Physiological stress induced by flickering light further influenced this. These results implicate stress-induced changes in the rate of transretinal fluid movement in the development of refractive error

Hyperosmotic stress and osmo-gene adaptation during early induction of refractive errors

Abstract Purpose: Why is myopia a common risk factor for most sight threatening disorders? Our earlier biometric, ultrastructural and elemental analyses of the chick form deprivation model have provided evidence of severe physiological, oxidative and hyperosmotic stress. More recently prolonged hyperosmotic stress has been shown to lead to chronic inflammation in a number of diseases (Brocker etal 2013). We hypothesized that perturbation of axial growth during induction of refractive errors would also be accompanied by hyperosmosis and osmoadaptative gene changes, that should be demonstratable with elemental microanalysis (EDX) and RNA seq respectively. Methods: Chicks were raised with ±10D lenses, or no lens. Following biometric measurements at 1, 2, and 3 days, 8 chicks per lens group were euthanized. RNA was extracted from the retina/RPE/choroid of 4. Four were used for scanning electron-microscopy and EDX. Libraries were sequenced on the Illumina HiSeq1500. Counts per million were imported into GSEA and expression of KEGG and Reactome pathways during myopia/hyperopia induction compared to age-matched no lens chicks (FDR cut-off <.25). Results: Refractive compensation (RC) to -10D defocus continued for 72hrs whereas RC to +10D was in near completion after 24hours. EDX shows sodium and chloride ion distributions were greatly upregulated in outer retina by -10D over the 72hrs but only at the retino-vitreal border in +10D at 72hrs. Potassium profiles in RC to +10D remained upregulated across the retina for 72 hrs with concurrent up-regulation of reactome potassium channel pathways at 72hrs in RNAseq data. Consistent with altered osmotic and oxidative stress, implicated pathways during refractive compensation included those related to synthesis of small molecule osmolytes, structural remodelling, inflammation, and metabolism. Conclusions: The EDX results demonstrate that RC to optical defocus is accompanied by hyperosmotic shifts in ion distribution profiles across the entire posterior eye, while concurrent changes in gene expression profiles were seen in metabolic and ion solute processes. These pathways have previously been associated with osmoadaptation and more severe disease states such as ARM and diabetes. The findings suggest the need for further experimental considerations of hyperosmotic changes as risk factors for severe visual impairments and for development of therapeutics

Spatial and temporal dissociation of AQP4 and Kir4.1 expression during induction of refractive errors

Purpose: Spatial co-localization of aquaporin water channels (AQP4) and inwardly rectifying potassium ion channels (Kir4.1) on the endfeet regions of glial cells has been suggested as the basis of functionally interrelated mechanisms of osmoregulation in brain edema. The aim of this study was to investigate the spatial and temporal changes in the expression of AQP4 and Kir4.1 channels in an avascular retina during the first week of the optical induction of refractive errors. Methods: Three-day-old hatchling chicks were randomly assigned to three groups and either did not wear lenses or were monocularly goggled with +/- 10D lenses for varying times up to 7 days before biometric assessment. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 and Kir4.1 protein in the chick retina or for immunolocalization using AQP4 and Kir4.1 antibodies to determine the regional distribution and intensity of labeling during the induction of refractive errors. Results: As expected, ultrasonography demonstrated that all eyes showed rapid elongation post hatching. Negative lens-wearing eyes elongated faster than fellow eyes or normal non goggled eyes and became progressively more myopic with time post lensing. Positive lens-wearing eyes showed reduced ocular growth compared to normal controls and developed a hyperopic refraction. Quantitative immunohistochemistry revealed the upregulation of AQP4 channel expression on Muller cells in the retinal nerve fiber layer during the first 2 days of negative lens wear. Kir4.1 channel upregulation in the inner plexiform layer was only found on day 4 of positive lens wear during the development of refractive hyperopia. Conclusions: These results indicate that the expression of AQP4 and Kir4.1 channels on Muller cells is associated with the changes in ocular volume seen during the induction of refractive errors. However, the sites of greatest expression and the temporal pattern of the upregulation of AQP4 and Kir4.1 were dissimilar, indicating a dissociation of AQP4 and Kir4.1 function during refractive error development. Increased AQP4 expression in the nerve fiber layer is suggested to contribute to the rapid axial elongation and movement of fluid into the vitreous cavity in the presence of minus lenses; whereas, upregulation of Kir4.1 channels appears to play a role in limiting axial elongation in the presence of plus lenses