Functional connexin35 increased in the myopic chicken retina.
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ABSTRACT:
SUBMITTER: Banerjee S
PROVIDER: S-EPMC8167454 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myopia is a substantial public health problem worldwide. In the myopic retina, distant images are focused in front of the photoreceptors. The cells and mechanisms for retinal signaling that account either for emmetropization (i.e., normal refraction) or for refractive errors have remained elusive. Gap junctions play a key component in enhancement of signal transmission in visual pathways. AII amacrine cells (ACs), coupled by connexin36, segregate signals into ON and OFF pathways. Coupling between AII ACs is actively modulated through phosphorylation at serine 293 via dopamine in the mouse retina. In this study, form deprivation mouse myopia models were used to evaluate the expression patterns of connexin36-positive plaques (structural assay) and the state of connexin36 phosphorylation (functional assay) in AII ACs, which was green fluorescent protein-expressing in the Fam81a mouse line. Single-cell RNA sequencing showed dopaminergic synapse and gap junction pathways of AII ACs were downregulated in the myopic retina, although Gjd2 mRNA expression remained the same. Compared with the normal refractive eye, phosphorylation of connexin36 was increased in the myopic retina, but expression of connexin36 remained unchanged. This increased phosphorylation of Cx36 could indicate increased functional gap junction coupling of AII ACs in the myopic retina, a possible adaptation to adjust to the altered noisy signaling status.
| S-EPMC7278884 | biostudies-literature
Project description:The retina plays an important regulatory role in ocular growth. To screen for new retinal candidate genes that could be involved in the inhibition of ocular growth, we used chick microarrays to analyze the changes in retinal mRNA expression after myopic defocus was imposed by positive lens-wear. Chicks were raised under a 12h-light/12h-dark cycle (light-onset: 8:00 am and light-offset: 8:00 pm) with unrestricted access to water and food. On the day prior to the experiment, velcro rings were glued to the feathers around the eye under diethylether anaesthesia. Four male white leghorn chicks, aged 9 days, wore +6.9D spectacle lenses over both eyes for 24 hours. Four untreated age-matched male chicks from the same batch served as controls. After decapitation of the chicks, the eyes were enucleated and the retina was prepared. The retinae from both eyes of each chick were pooled for RNA isolation. Experiment Overall Design: Four samples each: Retina of both eyes of +6.9 diopter lens-treated chicks (plus_lens), Retina of both eyes of untreated control chicks (control)
2008-11-04 | E-GEOD-11439 | biostudies-arrayexpress
Project description:RNA sequencing transcriptome analysis using massively parallel next generation sequencing technology provides the capability to understand global changes in gene expression throughout a range of tissue samples. Development of the vertebrate retina requires complex temporal orchestration of transcriptional activation and repression. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis. Existing retinal transcriptome projects have been critical to the vision research community for studying aspects of murine and human retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal transcriptome. Here we used Illumina RNA sequencing (RNA-seq) analysis to characterize the mRNA transcriptome of the developing chicken retina in an effort to identify genes critical for retinal development in this important model organism. These data will be valuable to the vision research community for characterizing global changes in gene expression between ocular tissues and critical developmental time points during retinogenesis in the chicken retina.
| S-EPMC5170595 | biostudies-literature
Project description:Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < -0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), "lipid metabolism" was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.
| S-EPMC8124159 | biostudies-literature
Project description:Chickens were kept under a regimen of 12 hours of light and 12 hours of dark for 7 days. At LD2, 2 hours into the light cycle on the 8th day, 3 chickens were sacrificed by decapitation and their retinas dissected and pooled. Similarly, at LD18, 6 hours into the dark cycle on the 8th day, retinas were harvested from 3 chickens. Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion). Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3. Hybridizations and washes were as suggested by Genisphere. Labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios.
2010-12-10 | GSE25992 | GEO
Project description:KCNQ5 is suggestively associated with myopia, but its specific role in the myopic process has not been studied further. The aim of this study was to investigate the expression of potassium channel gene KCNQ5 and the changes of K+ microenvironment within the retina of form deprivation myopia (FDM) guinea pigs. A total of 60 guinea pigs were randomly divided into the normal control (NC) group, the self-control (SC) group, and the form-deprivation (FD) group for different treatments. Molecular assays and immunohistochemistry (IHC) were conducted to measure the expression and distribution of KCNQ5-related gene and protein in the retina. We determined the K+ concentration in the retina. In addition, the possible effects of form deprivation on potassium ionic currents and the pharmacological sensitivity of KCNQ5 activator Retigabine and inhibitor XE991 to the M-current in RPE cells were investigated using the patch-clamp technique. As a result, FD eyes exhibited more myopic refraction and longer AL. The mRNA and protein levels of KCNQ5 significantly decreased in the FD eyes, but the K+ concentration increased. In addition, the M-type K+ current [IK(M)] density decreased in FD RPE cells, and were activated or inhibited in a concentration-dependent manner due to the addition of Retigabine or XE991. Overall, KCNQ5 was significantly downregulated in the retina of FD guinea pigs, which may be associated with the increasing K+ concentration, decreasing IK(M) density, and elongating ocular axis. It suggested that KCNQ5 may play a role in the process of myopia, and the intervention of potassium channels may contribute to the prevention and control of myopia.
| S-EPMC8733613 | biostudies-literature
Project description:PurposeTo investigate the role of peripheral refraction in children's myopization.MethodsThis 2-year study included 214 children (8-15 years old). Refraction across the retina (field of view: 60° × 36°) was measured with a custom-made aberrometer every year. Three datasets were established: dataset 1, 214 subjects from baseline to the first-year visit; dataset 2, 152 subjects from baseline to the second-year visit; and dataset 3, 59 initial emmetropes from baseline to the second-year visit. The baseline refraction of different retina regions was correlated with the central myopic shift, and was compared among groups with different levels of myopic shift.ResultsIn datasets 1 and 2, the refraction distribution across the retina was significantly different among the subjects who were initially emmetropes but not among those who were initially hyperopic or myopic. Refraction in the central vertical retina, especially in the superior retina (r = -0.5, P < 0.001), was significantly correlated with the myopic shift for emmetropes in that subjects with more relative myopia in the superior retina manifested greater central myopic shifts. In dataset 3, 21 subjects remained emmetropic after 2 years, 15 subjects became myopic at the 1-year visit, and 23 subjects became myopic at the 2-year visit. No difference was found for the relative peripheral refraction in all of the peripheral regions between the stage prior to and after the onset of myopia.ConclusionsRelative myopic defocus in the superior retina could be a predictor of central myopia shift. Changes in relative peripheral refraction are more likely a consequence of myopia progression rather than a cause.
| S-EPMC10117224 | biostudies-literature
Project description:Chickens were kept under a regimen of 12 hours of light and 12 hours of dark for 7 days. At LD2, 2 hours into the light cycle on the 8th day, 3 chickens were sacrificed by decapitation and their retinas dissected and pooled. Similarly, at LD18, 6 hours into the dark cycle on the 8th day, retinas were harvested from 3 chickens. Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion). Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3. Hybridizations and washes were as suggested by Genisphere. Labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.
2010-12-10 | E-GEOD-25992 | biostudies-arrayexpress
Project description:PurposeThe posthatching chicken is a valuable animal model for research, but molecular tools needed for altering its gene expression are not yet available. Our purpose here was to adapt the adeno-associated viral (AAV) vector method, used widely in mammalian studies, for use in investigations of the chicken retina. We hypothesized that the recently characterized avian AAV (A3V) vector could effectively transduce chick retinal cells for manipulation of gene expression, after intravitreal or subretinal injection.MethodsA3V encoding enhanced green fluorescent protein (EGFP) was injected intravitreally or subretinally into P1-3 chick eye and left for 7 to 10 days. Retinas were then sectioned or flat-mounted and visualized via laser-scanning confocal microscopy for analysis of expression and imaging of retinal cells.ResultsIntravitreal A3V-EGFP injection resulted in EGFP expression in a small percent of retinal cells, primarily those with processes and/or cell bodies near the vitreal surface. In contrast, subretinal injection of A3V-EGFP within confined retinal "blebs" produced high rates of transduction of rods and all types of cones. Some examples of all other major retinal cell types, including horizontal, amacrine, bipolar, ganglion, and Müller cells, were also transduced, although with much lower frequency than photoreceptors.ConclusionsA3V is a promising tool for investigating chick retinal cells and circuitry in situ. This novel vector can be used for studies in which local photoreceptor transduction is sufficient for meaningful observations.Translational relevanceWith this vector, the postembryonic chick retina can now be used for preclinical trials of gene therapy for prevention and treatment of human retinal disease.
| S-EPMC6608088 | biostudies-literature
Project description:Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (-15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of ?-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.
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