| Objectives:To induce experimental myopia by hyperopic defocus in mouse using a head-mounted spectacle frame apparatus, and assess the stability, security of apparatus and effectiveness of lens induction. Altered retinal miRNA expression profile was tested by microarray. Selected miRNAs underwent qRT-PCR to verify the concentration changes in myopic retina. Bioinformatics analysis was performed to reveal the relevant biological function of retina enriched miRNAs and experimental myopia differential expressed miRNAs.Methods:1. A head-mounted spectacle frame apparatus was designed and used in lens-induced myopia of C57BL6J mouse. The stability, security and effectiveness was assessed. The refraction of mouse eye was tested by streak retinoscopy. Axial parameters were measured with optical coherence tomography system, and the repeatability, reproducibility and accuracy was pre-experimental determined. All mice were divided into experimental group and control group, with an experimental period from ages of 4 weeks to 8 weeks. Myopia was monocularly induced over the right eye of the experimental mice using a-15.0 diopter (D) lens, while the contralateral eye and control group were subjected to no disposal.2. Microarray analysis using miRCURYTM LNA expression array (miRNA data source from miRBase 18.0) was performed to reveal the alteration of retinal miRNA expression in myopia. Concentration changes of selected miRNAs, including miR-328-3p,184-5p,183/96/182-5p,9-5p and181a-5, were verified by quantitative real-time PCR in both microarray samples and additional samples.3. Both retina enriched miRNAs and differential expressed miRNAs in myopia underwent bioinformatics analysis using various software and databases. The relevant biological function was elucidated mainly on validated target gene, predicated target gene and signing pathway involved.Results:1. In an effort to improve the available myopia induction device for mice, we described a novel apparatus named the head-mounted spectacle frame apparatus and represented a simplification of the currently employed head fixation techniques. Our results demonstrated numerous benefits, including improved compliance and decreased harmfulness and convenient fabrication. The optical coherence tomography system was built with high repeatability, reproducibility and accuracy. Refraction data demonstrated that the defocusing eye of the experimental mice achieved a significant myopic shift of -4.90±2.17 D (p<0.05) compared to the contralateral eye, and the defocusing eyes were significantly longer, mainly the vitreous body depth longer by 0.023±0.033mm (p<0.05). Both refraction and axial length were not significantly different between the right and left eyes of the control group (p>0.05).2. The miRNA expression profile of mouse retina was of tissue specificity. The miRNA expression profile of the myopia retina was significantly different from that of controls. However, the concentration changes were not as big as expected. A total of 71 miRNAs were upregulated by at least 1.2 folds, including miR-183/96/182-5p,29b-3p, 9-5p and 181a-5, etc.; 65 miRNAs were downregulated by at least 1.2 folds, including the let-7 family and miR-103-3p, etc.. With the quantitative real-time PCR technique, altered expression was verified in 5 of 7 selected miRNAs (miR-183/96/182-5p, 181a-5p and 9-5p), all of which were retina enriched.3. The tissue specific miRNA expression indicated that the retinal miRNAs play crucial role in physiological or pathological status. Bioinformatics analysis showed that the retina enriched miRNAs were primarily involved in synaptic function regulation, such as Dopaminergic synapse and Cholinergic synapse, as well as a number of experimental validated pathways, such as Insulin signaling pathway and Wnt signaling pathway. The altered miRNAs expressions in myopic retina, like miR-29 and let-7 families, were both involved in ECM-receptor interaction and PI3K-Akt signaling pathway.Conclusions:1. When a head-mounted spectacle frame apparatus was used for myopia induction in mouse, it showed good stability, safety, efficacy and promising for popularization. The customized built optical coherence tomography system was of good repeatability, reproducibility and accuracy. Hyperopic defocus induced myopia in mice under photopic conditions during the susceptible period in postnatal development. And the experimental myopia in mice is associated with elongation of the axial length of the eye.2. Altered retinal miRNA expression was found in experimental myopia with a slight extent. Among them there are 71 miRNA expression increased ≥1.2 times in myopic retina, including miR-183/96/182-5p,29b-3p,9-5p and 181 a-5p, etc; and 65 miRNA expression decreased ≥1.2 times, including miR- 98-5p, miR-103-3p and let-7 family. Real-time quantitative RCR verified the miR-183/96/182-5p,9-5p and 181a-5p expression in myopic retina was significantly higher than the self-control eye.3. The retinal enriched miRNAs played important role in physiological regulation and pathological mechanism. The altered miRNA expression profile of myopia retina was determined by microarray analysis, and partially verified with quantitative real-time PCR. Among which, miR-29 and let-7 families were both associated with functional regulation of extracellular matrix and PI3K-Akt signaling pathways, which may contribute to the lens-induced myopia. It was necessary and promising to focusing on these molecules and pathways in further study. |
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