GABAB Receptor Activation Affects Eye Growth in Chickens with Visually Induced Refractive Errors.

This study aims to explore the role of GABAB receptors in the development of deprivation myopia (DM), lens-induced myopia (LIM) and lens-induced hyperopia (LIH). Chicks were intravitreally injected with 25 µg baclofen (GABABR agonist) in one eye and saline into the fellow eye. Choroidal thickness (ChT) was measured via OCT before and 2, 4, 6, 8, 24 h after injection. ChT decreased strongly at 6 and 8 h after baclofen injection and returned back to baseline level after 24 h. Moreover, chicks were monocularly treated with translucent diffusers, -7D or +7D lenses and randomly assigned to baclofen or saline treatment. DM chicks were injected daily into both eyes, while LIM and LIH chicks were monocularly injected into the lens-wearing eyes, for 4 days. Refractive error, axial length and ChT were measured before and after treatment. Dopamine and its metabolites were analyzed via HPLC. Baclofen significantly reduced the myopic shift and eye growth in DM and LIM eyes. However, it did not change ChT compared to respective saline-injected eyes. On the other hand, baclofen inhibited the hyperopic shift and choroidal thickening in LIH eyes. All the baclofen-injected eyes showed significantly lower vitreal DOPAC content. Since GABA is an inhibitory ubiquitous neurotransmitter, interfering with its signaling affects spatial retinal processing and therefore refractive error development with both diffusers and lenses.

Effects of Single and Repeated Intravitreal Applications of Atropine on Choroidal Thickness in Alert Chickens.

INTRODUCTION: Atropine, a muscarinic antagonist, is known since the 19th century to inhibit myopia development in children. One of its effects is that it stimulates choroidal thickening. Thicker choroids, in turn, have been linked to myopia inhibition. We used the atropine-stimulated choroidal response in the chicken to learn more about the time courses and amplitudes of the effects of atropine, as well as whether repeated applications lead to accumulation or desensitization. METHODS: Intravitreal injections containing 250 µg atropine sulfate were performed in 1 eye around 10:00 in the morning, the fellow eye received vehicle. Chickens with bilateral vehicle injections served as controls. Choroidal thickness was measured over the day for every 2-3 h in alert animals, using spectral domain optical coherence tomography, with 3-5 independent measurements in each eye. Three experiments were done - (1) single injection and time course measured over 1 day, (2) single injection and time course measured over 4 days, and (3) daily injections and time course measured over 4 days for measuring the effects of atropine on vitreal, retinal, and choroidal dopamine, and 3,4-dihydroxyphenylacetic acid levels by using high-performance liquid chromatography with electrochemical detection. RESULTS: Atropine induced an increase in choroidal thickness by about 60 percent, with a peak amplitude after about 2 h. The effect persisted only for a few hours and had nearly disappeared by evening. Initially, similar amounts of choroidal thickening were observed in vehicle-injected fellow eyes but recovery to baseline was faster. When atropine was injected daily for 4 days, choroids thickened every day with similar amplitudes and time courses, with no signs of either accumulation or desensitization effects. Interestingly, while dopamine release from the retina was stimulated by atropine and followed approximately, the time course of choroidal thickening, its tissue concentration dropped in the choroid. CONCLUSIONS: Even at relatively high intravitreal doses, effects of atropine on choroidal thickness remained transient, similar to its effects on retinal dopamine. With repeated application every day, the diurnal patterns of choroidal thickening could be reproduced for 4 days with similar amplitudes and time courses. The transient nature of the effects of atropine on the choroid may be relevant for application protocols of atropine against myopia.

Gene expression within the amacrine cell layer of chicks after myopic and hyperopic defocus.

PURPOSE. Ocular growth is regulated locally by signals produced in the retina. The highly heterogeneous nature of the retina may mask important changes in gene expression during global analysis. This study was conducted to investigate changes in gene expression specifically within the amacrine cell layer (ACL), the most likely generator of growth signals, during optical manipulation of ocular growth. METHOD. Chicks were monocularly treated with either -7-D (n = 6) or +7-D (n = 6) lenses for 24 hours. Untreated age-matched chicks served as control subjects (n = 6). Total RNA from the ACL was isolated from 10-mum-thick sections, obtained using laser capture microdissection. Labeled cRNA was prepared from three samples per condition and hybridized to chicken genome microarrays. Changes in gene expression were validated by using semiquantitative real-time RT-PCR. RESULTS. One hundred twenty-eight genes were differentially expressed in the ACL of the minus lens-treated eyes, whereas the plus lens-treated eyes displayed 58 changes 24 hours after treatment. Only 11 genes were differentially expressed under both experimental conditions, whereas the expression of only one gene (clone ChEST927g14) was modulated by the sign of defocus. Compared with previous studies in the field, the magnitude of changes observed in the present work were larger, with more than 30% of differentially expressed genes showing a twofold or greater modulation in expression. The results, obtained from independent validation by real-time RT-PCR technology, correlated highly with the original microarray data. The differential expression of four of eight genes was validated in plus lens-treated eyes, and eight of nine genes were independently validated in minus lens-treated eyes. CONCLUSIONS. The targeted investigation of the ACL enabled the identification of several novel genes that may form part of the growth regulatory pathways of the eye. Different retinal pathways may underlie the response of the eyes to plus and minus lens compensation, as there was limited overlap in the regulated genes observed within the ACL under both conditions.

Microarray analysis of retinal gene expression in Egr-1 knockout mice.

PURPOSE: We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days. METHODS: The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT-PCR. RESULTS: Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT-PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT-PCR. Changes in four of the ten genes could be confirmed by real-time RT-PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others. CONCLUSIONS: Identification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study.

Microarray analysis of retinal gene expression in chicks during imposed myopic defocus.

PURPOSE: 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. METHODS: Four male white leghorn chicks, aged nine days, wore +6.9D spectacle lenses over both eyes for 24 h. Four untreated age-matched male chicks from the same batch served as controls. The chicks were euthanized, and retinas from both eyes of each chick were pooled. RNA was isolated and labeled cRNA was prepared. These samples were hybridized to Affymetrix GeneChip Chicken Genome arrays with more than 28,000 characterized genes. After comparison of multiple normalization methods, GC-RMA and a false-discovery rate of 6% was chosen for normalization of the data. The expression of 16 candidate genes was further studied, using semiquantitative real-time RT-PCR. In addition, the expression of the mRNA of some of these candidate genes was assessed in chicks that wore either +6.9D lenses for 4 h or -7D lenses for 24 h. RESULTS: 123 transcripts were found to be differentially expressed (p<0.05; at least 1.5-fold change in expression level), with an absolute mean fold-change of 1.97+/-1.16 (mean+/-standard deviation). Nine of the sixteen genes that were examined by real-time RT-PCR were validated. Regardless of whether positive or negative lenses were worn, six of these nine genes were regulated in the same direction after 24 h: arginyltransferase 1 (ATE1), E74-like factor 1 (ELF1), growth factor receptor-bound protein 2 (GRB2), SHQ1 homolog (S. cerevisiae) (SHQ1), spectrin, beta, non-erythrocytic 1 (SPTBN1), prepro-urotensin II-related peptide (pp-URP). Three genes responded differently to positive and negative lens treatment after 24 h: ATP-binding cassette, sub-family C, member 10 (ABCC10), CD226 molecule (CD226) and oxysterol binding protein 2 (OSBP2). CONCLUSIONS: The validated genes that were regulated only by myopic defocus may represent elements in a pathway generating a "stop-signal" for eye growth. Some of the genes identified in this study have so far not been described in the retina. Further investigation of their function may improve the understanding of the signaling cascades in emmetropization. More general, published microarray data are variable among different animal models (mouse, chick, monkeys), tissues (retina, retina/retinal pigment epithelium), treatments (diffusers, lenses, lid-suture), as well as different treatment durations (hours, days), and comparisons remain difficult. That only a small number of common genes were found emphasizes the need for careful normalization of the experimental parameters.

A microarray analysis of retinal transcripts that are controlled by image contrast in mice.

PURPOSE: The development of myopia is controlled by still largely unknown retinal signals. The aim of this study was to investigate the changes in retinal mRNA expression after different periods of visual deprivation in mice, while controlling for retinal illuminance. METHODS: Each group consisted of three male C57BL/6 mice. Treatment periods were 30 min, 4 h, and 6+6 h. High spatial frequencies were filtered from the retinal image by frosted diffusers over one eye while the fellow eyes were covered by clear neutral density (ND) filters that exhibited similar light attenuating properties (0.1 log units) as the diffusers. For the final 30 min of the respective treatment period mice were individually placed in a clear Perspex cylinder that was positioned in the center of a rotating (60 degrees) large drum. The inside of the drum was covered with a 0.1 cyc/degree vertical square wave grating. This visual environment was chosen to standardize illuminances and contrasts seen by the mice. Labeled cRNA was prepared and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Alterations in mRNA expression levels of candidate genes with potential biological relevance were confirmed by semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In all groups, Egr-1 mRNA expression was reduced in diffuser-treated eyes. Furthermore, the degradation of the spatial frequency spectrum also changed the cFos mRNA level, with reduced expression after 4 h of diffuser treatment. Other interesting candidates were Akt2, which was up-regulated after 30 min of deprivation and Mapk8ip3, a neuron specific JNK binding and scaffolding protein that was temporally regulated in the diffuser-treated eyes only. CONCLUSIONS: The microarray analysis demonstrated a pattern of differential transcriptional changes, even though differences in the retinal images were restricted to spatial features. The candidate genes may provide further insight into the biochemical short-term changes following retinal image degradation in mice. Because deprivation of spatial vision leads to increased eye growth and myopia in both animals and humans, it is believed some of the identified genes play a role in myopia development.

Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

PURPOSE: To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. METHODS: Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. RESULTS: The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. CONCLUSIONS: Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.