MicroRNA-152-3p and MicroRNA-196a-5p Are Downregulated When Müller Cells Are Promoted by Components of the Internal Limiting Membrane: Implications for Macular Hole Healing.

Müller cells play a critical role in the closure of macular holes, and their proliferation and migration are facilitated by the internal limiting membrane (ILM). Despite the importance of this process, the underlying molecular mechanism remains underexplored. This study investigated the effects of ILM components on the microRNA (miRNA) profile of Müller cells. Rat Müller cells (rMC-1) were cultured with a culture insert and varying concentrations of ILM component coatings, namely, collagen IV, laminin, and fibronectin, and cell migration was assessed by measuring cell-free areas in successive photographs following insert removal. MiRNAs were then extracted from these cells and analyzed. Mimics and inhibitors of miRNA candidates were transfected into Müller cells, and a cell migration assay and additional cell viability assays were performed. The results revealed that the ILM components promoted Müller cell migration (p < 0.01). Among the miRNA candidates, miR-194-3p was upregulated, whereas miR-125b-1-3p, miR-132-3p, miR-146b-5p, miR-152-3p, miR-196a-5p, miR-542-5p, miR-871-3p, miR-1839-5p, and miR-3573-3p were significantly downregulated (p < 0.05; fold change > 1.5). Moreover, miR-152-3p and miR-196a-5p reduced cell migration (p < 0.05) and proliferation (p < 0.001), and their suppressive effects were reversed by their respective inhibitors. In conclusion, miRNAs were regulated in ILM component-activated Müller cells, with miR-152-3p and miR-196a-5p regulating Müller cell migration and proliferation. These results serve as a basis for understanding the molecular healing process of macular holes and identifying potential new target genes in future research.

Circulating Vitreous microRNA as Possible Biomarker in High Myopic Eyes with Macular Hole.

High myopia is a major cause of irreversible visual impairment globally. In the present study, we investigated the microRNA (miRNA) profile in the vitreous of macular hole (MH) and high myopic MH. We performed miRNA analysis using TaqMan® Low Density Arrays (Thermo Fisher Scientific, Waltham, MA, USA) to investigate the circulating vitreous miRNA profile from patients with MH (axial length < 26.5 mm, n = 11) and high myopic MH (axial length ≥ 26.5 mm, n = 11) who underwent pars plana vitrectomy. The vitreous inflammatory cytokine signature was examined in high myopic MH eyes using a multiplex assay. A miRNA-Array analysis revealed that let-7c was significantly up-regulated and miR-200a was significantly down-regulated in high myopic MH eyes compared to those in MH eyes. The bioinformatics analysis for up-regulated miRNA targeted gene identified 23 pathways including mitogen-activated protein kinase (MAPK) and several inflammatory signaling pathways, whereas the bioinformatics analysis for down-regulated miRNA targeted genes showed 32 enriched pathways including phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). The levels of inflammatory cytokines including IP-10, IFN-γ, and MCP-1 were significantly higher in the vitreous of high myopic MH eyes. These results suggest that specific miRNAs expressed in the vitreous may be associated with the pathological condition of high myopic MH and the above mentioned miRNAs may contribute to the development of inflammatory status in the vitreous of high myopic eyes.

Correlations between vitreous cytokine levels and inflammatory cells in fibrovascular membranes of patients with proliferative diabetic retinopathy.

Purpose: The purpose of this study was to investigate the levels of cytokines in the vitreous, and their correlation with the density of inflammatory cells in fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR) to evaluate intraocular inflammatory conditions with regard to disease activity. Methods: Thirty-three patients (33 eyes) with PDR requiring vitreoretinal surgery because of FVMs and tractional detachment were enrolled in the study, and compared with 20 patients (20 eyes) with macular hole (MH; control group). All patients underwent complete ophthalmological examinations before surgery. The activity of the disease was noted in patients with PDR. Samples of vitreous and blood were taken, and cytokine (MCP-1, IL-8, IL-6, VEGF, IL-1ß, TNF-α, MIP-1α, MIP-1ß, IL-10, and IL-12) levels were measured using cytometric bead array (CBA). Samples of FVMs were analyzed with immunohistochemical methods for the presence of inflammatory cells (CD45+, CD14+, CD3+, CD4+, CD8+, and CD19+ cells), and the numerical areal density was calculated (NA). Spearman's correlation was used to assess the association between variables. The Mann-Whitney test was used to assess the differences between independent groups. The Wilcoxon signed-rank test was used for assessing differences between two related groups. A p value of less than 0.05 was considered statistically significant. Results: Patients with active PDR had statistically significantly higher levels of MCP-1 (p = 0.003), VEGF (p = 0.009), and IL-8 (p = 0.02) in the vitreous in comparison with those with inactive PDR. CD45+, CD14+, CD3+, CD4+, CD8+, and CD19+ cells were identified in FVMs for patients with PDR. Statistically significantly higher numerical areal density of T lymphocytes (CD3+, CD4+, and CD8+) was demonstrated in patients with active PDR in comparison with patients with inactive PDR. Moderate to strong correlations were found between either MCP-1 or IL-8 in the vitreous, and the numerical areal density of cells (CD45+, CD3+, CD4+, and CD8+) in the FVMs, and weaker between either MCP-1 or IL-8 in the vitreous and the numerical areal density of CD14+ cells in the FVMs. Conclusions: The correlation of cytokine (MCP-1 and IL-8) vitreous levels with the density of inflammatory cells in FVMs, and differences in cytokine levels in the vitreous between patients with active and inactive PDR, and between the vitreous and serum in PDR indicate the importance of local intraocular inflammation in patients with PDR.

Bilateral giant retinal tears in Osteogenesis Imperfecta.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare primarily autosomal dominant condition in which the connective tissues of bones, ligaments and sclerae do not form properly. Typically, mutations in COL1A1 and COL1A2 genes lead to the defective formation or quantity of type I collagen, the principle matrix in these tissues. Molecular genetic studies have now elucidated multiple genetic subtypes of the disorder but little literature exists on the risk of retinal tears and detachments in OI. CASE PRESENTATION: We report the first case of a child with a rare recessive type of OI, subtype VIII, resulting from a P3H1 (also known as LEPRE1) gene mutation presenting with bilateral giant retinal tears and the surgical challenges encountered in performing retinal detachment repair due to scleral thinning. The P3H1 gene encodes for prolyl 3-hydroxylase 1 which is involved in the post-translational modification of not only collagen type I but also types II and V which when mutated may result in pathological posterior vitreous detachment (PVD) and giant retinal tear detachments. CONCLUSIONS: Genetic analyses are increasingly important in such cases and may guide patient monitoring and potential prophylactic treatment, known to significantly reduce the probability of giant retinal tear detachments in other high-risk collagenopathies such as Stickler Syndrome Type I.

Comparisons of microRNA expression profiles in vitreous humor between eyes with macular hole and eyes with proliferative diabetic retinopathy.

PURPOSE: MicroRNAs (miRNAs) are small noncoding RNAs which regulate the activities of target mRNAs. We compared the expression profiles of the miRNAs in the vitreous of eyes with macular hole (MH) to that in eyes with proliferative diabetic retinopathy (PDR). METHODS: Vitreous and whole blood samples were collected from four patients with MH and from four patients with PDR. We assayed for 168 miRNAs in the vitreous and serum samples by the microRNA PCR Panel method. RESULTS: The mean number of miRNAs expressed in the vitreous was 63 (55-69) in eyes with MH and 86 (65-117) in eyes with PDR. The mean number of miRNAs expressed in the serum was 162 (159-167) in the MH patients and 142 (115-160) in the PDR patients. Twenty-six miRNAs were expressed in the vitreous of both MH and PDR eyes. Although there was no significant difference in the levels of 20 of the 26 (73 %) miRNAs expressed in both MH and PDR eyes, six of 26 miRNAs (24 %) (hsa-miR-15a, hsa-miR320a, hsa-miR-320b, hsa-miR-93, hsa-miR-29a, and hsa-miR-423-5p) were expressed significantly more highly in PDR eyes. In addition, the mean fold changes of three miRNAs, hsa-miR-23a, hsa-miR-320a, and hsa-miR-320b, in the vitreous to serum were significantly higher in the PDR group than in the MH group. CONCLUSIONS: The expression of several miRNAs related to angiogenesis and fibrosis was expressed significantly higher in the vitreous of eyes with PDR. Further studies are needed to understand the role played by the miRNAs in the biological function of the eye.

Myopic Macular Hole and Detachment after Gene Therapy in Atypical RPE65 Retinal Dystrophy: A Case Report.

PURPOSE: To report a case of macular hole and detachment occurring after the subretinal injection of Voretigene Neparvovec (VN) in a patient affected by atypical RPE65 retinal dystrophy with high myopia and its successful surgical management. CASE DESCRIPTION: We report a case of a 70-year-old man treated with VN in both eyes. The best corrected visual acuity (BCVA) was 0.7 LogMar in the right eye (RE) and 0.92 LogMar in the left eye (LE). Axial length was 29.60 mm in the RE and 30.28 mm in the LE. Both eyes were pseudophakic. In both eyes, fundus examination revealed high myopia, posterior staphyloma, and extended retinal atrophy areas at the posterior pole, circumscribing a central island of surviving retina. Both eyes were treated with VN subretinal injection, but a full-thickness macular hole and retinal detachment occurred in the LE three weeks after surgery. The patient underwent 23-gauge vitrectomy with internal limiting membrane (ILM) peeling and the inverted flap technique with sulfur hexafluoride (SF6) 20% tamponade. Postoperative follow-up showed that the macular hole was closed and the BCVA was maintained. CONCLUSIONS: Our experience suggests that patients with atypical RPE65 retinal dystrophy and high myopia undergoing VN subretinal injection require careful management to minimize the risk of macular hole and detachment occurrence and promptly detect and address these potential complications.

MicroRNAs in vitreus humor from patients with ocular diseases.

PURPOSE: Based on evidence that microRNAs (miRNAs) are found in many biologic fluids (e.g., urine, saliva, pleural fluid), we sought to detect the presence of miRNAs and analyze their profile in vitreous humor (VH) from patients affected by various ocular diseases. METHODS: MiRNAs were purified from VH samples taken during vitrectomy, by using the Qiagen miRNeasy Mini Kit. The expression profile on 745 miRNAs was performed by using TaqMan Low Density Array. Single TaqMan expression assays were performed on 18 VH samples (six each from patients with choroidal melanomas, retinal detachment, or macular hole) for miRNAs commonly expressed in serum or retinal cells: let-7b, miR-21, miR-26a, miR-146a, miR-199-3p, miR-210, miR-374a*, miR-532-5p. RNA extracted from serum of six healthy donors or from formalin-fixed, paraffin-embedded samples of choroidal melanocytes from four uveal melanomas (epithelioid cells) and from three unaffected eyes were used as controls. RESULTS: We identified the presence of 94 circulating small RNAs in the vitreous, some of which (miR-9, miR-9*, miR-125a-3p, miR-184, miR-211, miR-214, miR-302c, miR-452, miR-628, miR-639) are particularly abundant in the VH but downrepresented or not detectable in serum. MiR-146a and miR-26a were overexpressed more than threefold in VH from patients with uveal melanomas compared to the other pathological groups (Wilcoxon signed-rank test, p value < 0.05). CONCLUSIONS: Our experimental data suggest that a specific set of circulating miRNAs is secreted in the vitreous, which is quite different from the miRNA pattern in serum, and that the quantity of vitreal miRNAs could change, depending on the pathologies of the eye.

Prevalence, Concordance, and Heritability of Vitreomacular Interface Abnormalities in a Twin Study.

Purpose: The relative importance of genetic factors in common vitreomacular interface (VMI) abnormalities is unknown. The aim of this classical twin study is to determine the prevalence case wise concordance between monozygotic and dizygotic twin pairs, and heritability of common VMI abnormalities, including epiretinal membrane (ERM), posterior vitreous detachment (PVD), vitreomacular adhesion (VMA), vitreomacular traction (VMT), lamellar macular holes (LMHs), and full-thickness macular holes (FTMHs). Methods: This is a single-center, cross-sectional classical twin study of 3406 TwinsUK participants over the age of 40 years who underwent spectral domain macular optical coherence tomography (SD-OCT) scans which were graded for signs of VMI abnormalities. Case wise concordance was calculated and the heritability of each VMI abnormality was estimated using OpenMx structural equation modeling. Results: In this population (mean age = 62.0 years [SD = 10.4 years], range = 40-89 years) the overall prevalence of ERM was 15.6% (95% confidence interval [CI] = 14.4-16.9) and increased with age, posterior vitreous detachment affected 21.3% (20.0-22.7), and VMA was diagnosed in 11.8% (10.8-13.0). Monozygotic twins were more concordant for all traits than dizygotic twins, and age, spherical equivalent refraction (SER), and lens status-adjusted heritability was estimated at 38.9% (95% CI = 33.6-52.8) for ERM, 53.2% (95% CI = 41.8-63.2) for PVD, and 48.1% (95% CI = 33.6-58) for VMA. Conclusions: Common VMI abnormalities are heritable and therefore have an underlying genetic component. Given the sight-threatening potential of VMI abnormalities, further genetic studies, such as genomewide association studies, would be useful to identify genes and pathways implicated in their pathogenesis.

Familial trends in a population with macular holes.

PURPOSE: To determine if patients with macular hole report an increased family history of macular hole compared with control patients and compare the report of family history between patients with unilateral and bilateral macular holes. METHODS: This was a multicenter case-control study. Charts of patients coded with diagnosis of macular hole were reviewed, and the diagnosis of idiopathic full-thickness macular hole was ascertained in 166 patients. The control group comprised 136 patients without macular hole or trauma who presented with senile cataract. Family history was obtained from all patients through a telephone interview. RESULTS: Six of 166 (3.6%) macular hole patients surveyed reported a history of macular hole in a primary relative compared with none of 136 (0.0%) control patients (odds ratio is infinity, with 95% confidence interval 1.295 to infinity); however, this finding may be explained by confounders such as age and number of family members. Two of the 142 (1.4%) patients with unilateral holes versus 4 of the 24 (16.7%) patients with bilateral holes reported a family history (odds ratio is 0.0714, with 95% confidence interval 0.0063 to 0.5537), and this finding remains significant when logistic regression is performed to evaluate variables of age and number of family members as potential confounders. CONCLUSION: There is an increased report of familial occurrence of macular hole in patients with macular holes compared with control patients; however, logistic regression relates this finding to variables of age and number of family members. Patients with bilateral macular holes are more likely to report a family history of macular hole than patients with unilateral macular holes, and this finding remains significant in the presence of age and number of family members. These findings may suggest a familial component to macular hole.

Impact of interleukin-6 promoter polymorphism and serum interleukin-6 level on the acute inflammation and neovascularization stages of patients with Eales' disease.

PURPOSE: To evaluate the role of interleukin-6 (IL-6) in the inflammatory and proliferative stages of Eales' disease (ED) and to determine the influence of IL-6-174G/C polymorphism in the IL-6 and IL-6-regulated protein expression, as well as the development of ED. METHODS: One hundred and twenty-one patients diagnosed with ED, 223 matched healthy controls, and 16 control patients with macular holes were recruited from the eastern Indian population. Serum and vitreous levels of IL-6 and vascular endothelial growth factors (VEGF) were measured by enzyme-linked immunosorbent assay. Serum levels of high-sensitivity C-reactive protein (hsCRP) were measured by enzyme immunoassay. Subjects were genotyped for the IL-6-174G/C polymorphism (rs1800795) by a custom TaqMan single-nucleotide polymorphism (SNP) Genotyping Assays system. RESULTS: Serum IL-6 (p<0.0001), hsCRP (p<0.0001), and VEGF (p=0.0031) levels were significantly higher in the inflammatory stage of ED than in healthy controls. Serum IL-6 also significantly correlated with hsCRP (Spearman's correlation coefficient; r=0.4992, p=0.0009), but not with VEGF in this stage in ED patients. At the proliferative stage of ED, significantly higher levels of vitreous IL-6 (p=<0.0001) and VEGF (p=<0.0001) were found compared with the vitreous of patients with macular holes. A significant correlation was observed between vitreous IL-6 and VEGF in ED patients (Spearman's correlation coefficient; r=0.5834, p=0.0087). A statistically significant association was found between the -174GG genotype (p=0.006) and occurrence of ED. Mean serum and vitreous concentrations of IL-6 were also higher in the subjects with the GG genotype than in those with the GC or CC genotype in this population. CONCLUSIONS: IL-6 expression, regulated by the allelic distribution of -174 loci and the enhanced level of IL-6, modulates CRP and VEGF concentration depending respectively on the acute inflammatory stimulation at the initial stage and angiogenic stimulation at the advanced stage of ED.

The predisposing pathology and clinical characteristics in the Scottish retinal detachment study.

PURPOSE: To describe the predisposing pathology and clinical features of all incident cases of rhegmatogenous retinal detachment (RRD) recruited in Scotland during a 2-year period. DESIGN: Prospective surveillance study of incident cases of RRD. PARTICIPANTS: All incident cases of RRD recruited as part of the Scottish Retinal Detachment Study. METHODS: During a 2-year period, we coordinated a comprehensive system in which every case of primary RRD presenting to 1 of 6 vitreoretinal surgical sites in Scotland was examined and approached for study inclusion. MAIN OUTCOME MEASURES: Rhegmatogenous retinal detachment incidence, predisposing features, and clinical characteristics. RESULTS: A total of 1202 cases were recruited. Detailed clinical information was available on 1130 (94%) of cases. By causative break, the proportions of RRD were horseshoe tear (HST) associated with posterior vitreous detachment (PVD) in 86.2%, giant retinal tear (GRT) and PVD in 1.3%, non-PVD round hole (RH) in 4.9%, retinal dialysis in 5.9%, and retinoschisis RRD in 1.6%. One in 10 cases reported significant ocular trauma. One in 5 cases were pseudophakic. Round hole RRD more frequently presented with multiple retinal breaks compared with HST RRD (67.8% vs. 48.7%; P = 0.003). In PVD-associated RRD, 56.1% (95% confidence interval [CI], 53.8-58.3) of breaks were identified in the superotemporal retina. In non-PVD RRD, 54.6% (95% CI, 47.9-61.1) of breaks were inferotemporal, followed by superotemporal in 34.9% (95% CI, 28.7-41.5). Lattice degeneration was present in 18.7% of affected eyes and more common in RH RRD (35.7%) than in HST RRD (19.3%) (P = 0.003). Seven percent reported an affected first-degree relative, and these cases were significantly more myopic than nonfamilial cases. CONCLUSIONS: More than 85% of RRD cases are associated with PVD and related tractional tears. Non-PVD RH RRD occurred in younger and more myopic individuals. The majority of cases are caused by more than 1 retinal break, and the macula is affected in more than 50% at presentation. Ocular trauma, previous cataract surgery, family history, and lattice degeneration are important predisposing features.

Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy.

AIMS/HYPOTHESIS: Interphotoreceptor retinoid-binding protein (IRBP) plays a major role in the visual cycle and is essential to the maintenance of photoreceptors. The aim of this study was to determine whether a decrease in IRBP production exists in the early stages of diabetic retinopathy. METHODS: Vitreous samples from diabetic patients with proliferative and non-proliferative diabetic retinopathy (PDR, NPDR), and from non-diabetic patients with macular hole (control group) were selected for IRBP quantitative assessment by proteomic analysis (fluorescence-based difference gel electrophoresis) and western blot. Human post mortem eyes (n = 16) from diabetic donors without clinically detectable retinopathy and from non-diabetic donors (n = 16) were used to determine IRBP (also known as RBP3) mRNA levels (RT-PCR) and protein content (western blot and confocal microscopy). Retinal neurodegeneration was assessed by measuring glial fibrillar acidic protein (GFAP) and the apoptotic rate. Y79 human retinoblastoma cells were used to test the effects of glucose, TNF-alpha and IL-1beta on IRBP expression and IRBP levels. RESULTS: Intravitreous IRBP concentration was significantly lower in PDR < NPDR < control in proteomic and western blot analysis. IRBP mRNA levels and IRBP protein content were significantly lower in the retinas from diabetic donors than in those from non-diabetic donors. Increased GFAP and a higher degree of apoptosis were observed in diabetic retinas compared with non-diabetic retinas. A dose-dependent downregulation of IRBP mRNA expression and IRBP content was detected with glucose, TNF-alpha and IL-1beta in cultures of Y79 human retinoblastoma cells. CONCLUSIONS/INTERPRETATION: Underproduction of IRBP is an early event in the human diabetic retina and is associated with retinal neurodegeneration. The mechanisms leading to this deficit deserve further investigation.

Analysis of the vitreous membrane in a case of type 1 Stickler syndrome.

BACKGROUND: Stickler syndrome causes ocular abnormalities, including retinal detachment and vitreoretinal degeneration, and systemic anomalies such as arthritis and deafness. Although retinal detachment is characteristic of this syndrome, the pathogenesis is unknown. CASE REPORT: A 10-year-old boy reported decreased vision and presented 5 days after visual loss. RESULTS: Ophthalmoscopy showed a retinal detachment with a giant tear in the right eye, and a nonpigmented epithelial detachment with pars plicata breaks in the left eye. Bilateral findings included an empty vitreous and a vitreous membrane at the equator. The systemic abnormalities included short stature and joint hypermobility. The diagnosis was type 1 Stickler syndrome, and the eyes were treated surgically. Immunohistochemistry showed that the vitreous membrane resected intraoperatively was comprised primarily of Müller cells. Electron microscopy showed dense collagen fibers around the cells in the membrane that were identical to the vitreous collagen inserted into the basement membrane of the cells, which was similar to the ultrastructure of the vitreous base. CONCLUSION: Müller cells might be primary components of the vitreous membrane in type 1 Stickler syndrome. The vitreoretinal interface, which resembled the ectopic vitreous base, in the vitreous membrane may be related to the pathogenesis of the retinal detachment.

miRNAs in the vitreous humor of patients affected by idiopathic epiretinal membrane and macular hole.

PURPOSE: The aim of the present study was to assess the expression of miRNAs in the Vitreous Humor (VH) of patients with Macular Hole (MH) and Epiretinal Membrane (ERM) compared to a control group. METHODS: In this prospective, comparative study, 2-ml of VH was extracted from the core of the vitreous chamber in consecutive patients who underwent standard vitrectomy for ERM and MH. RNA was extracted and TaqMan® Low Density Arrays (TLDAs) were used to profile the transcriptome of 754 miRNAs. Results were validated by single TaqMan® assays. Finally, we created a biological network of differentially expressed miRNA targets and their nearest neighbors. RESULTS: Overall 10 eyes with MH, 16 eyes with idiopathic ERM and 6 controls were enrolled in the study. Profiling data identified 5 miRNAs differentially expressed in patients affected by MH and ERM with respect to controls. Four were downregulated (miR-19b, miR-24, miR-155, miR-451) and 1 was downregulated (miR-29a); TaqMan® assays of the VH of patients affected by MH and ERM, with respect to controls, showed that the most differentially expressed were miR-19b (FC -9.13, p:<0.00004), mir-24 (FC -7.52, p:<0.004) and miR-142-3p (FC -5.32, p:<0.011). Our network data showed that deregulation of differentially expressed miRNAs induces an alteration of several pathways associated with genes involved in both MH and ERM. CONCLUSION: The present study suggests that disregulation of miR-19b, miR-24 and miR-142-3p, might be related to the alterations that characterize patients affected by MH and ERM.

An evolutionary perspective on the photoreceptor damage response.

PURPOSE: To review recent advances related to the response to photoreceptor damage and to place this knowledge in an evolutionary context. DESIGN: Synthesis of published laboratory, clinical, and epidemiologic data. METHODS: The authors have synthesized the principal published findings related to the mechanism and function of the photoreceptor damage response with the goal of trying to understand the selective pressures that shaped its evolution. RESULTS: The past several years have seen considerable advances in understanding the molecular and cellular basis of the retina's response to photoreceptor damage. From their analysis of laboratory, clinical, and epidemiologic data, the authors suggest that the photoreceptor damage response may have evolved to counter the effects of retinal tears and detachment, infectious retinitis, and/or light damage. CONCLUSIONS: If the natural response to photoreceptor damage can be fully defined, it may be possible to augment desirable aspects of the damage response and/or suppress undesirable ones in the context of a wide variety of photoreceptor diseases.

Gene Expression Analysis of the Irrigation Solution Samples Collected during Vitrectomy for Idiopathic Epiretinal Membrane.

PURPOSE: The analysis of gene expression in idiopathic epiretinal membranes (iERMs) may help elucidate ERM formation and its pathology. Here, we conducted a case-control study, in order to determine the expression levels of cytokines and other genes in eyes with macular hole (MH) or iERM. METHODS: Twenty eyes, obtained from seven male and 13 female patients, were included in the study. The average age of the study subjects was 69.1 ± 7.67 years, and 15 eyes had iERM, while five eyes had MH. Irrigation solution samples were collected during vitrectomy, centrifuged, and the levels of cytokine and other mRNAs in the sediment were assessed using real-time PCR. The expression level of 11 cytokine genes, four transcription factor genes, two cytoskeletal genes, and genes encoding two extracellular matrix proteins in eyes with MH or iERM were determined and compared. RESULTS: The expression levels of interleukin 6 (IL6), tumor growth factor B2 (TGFB2), vascular endothelial growth factor A (VEGFA), chemokine C-X-C motif ligand 1 (CXCL1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), glial fibrillary acidic protein (GFAP), and tenascin C (TNC) were significantly higher in eyes with iERM than in eyes with MH. The expression of these genes was not associated with the preoperative visual acuity of the investigated patients. CONCLUSIONS: The obtained results indicate that real-time PCR analysis of irrigation solution samples collected during vitrectomy can help assess the expression levels of several genes, and that iERM is associated with the expression of pro-inflammatory genes and the genes expressed during angiogenesis and wound healing process (IL6, TGFB2, VEGFA, CXCL1, RELA, GFAP, and TNC).

Th17 cell frequency and IL-17A concentrations in peripheral blood mononuclear cells and vitreous fluid from patients with diabetic retinopathy.

Objective To quantify T helper (Th)17 cells and determine interleukin (IL)-17A levels in peripheral blood mononuclear cell (PBMC) culture and vitreous fluid from patients with type 2 diabetes mellitus (T2DM) with diabetic retinopathy (DR). Methods Th17 cell frequency and IL-17A concentrations in PBMCs from 60 patients with T2DM with DR, 30 without DR and 30 sex- and age-matched healthy individuals were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. IL-17A levels in vitreous fluid from 31 eyes with proliferative DR and diabetic macular oedema (DR group) and 32 eyes with an epiretinal membrane and macular hole (control group) that underwent vitrectomy were also examined by ELISA. Results Compared with the control group, the proportion of Th17 cells and IL-17A concentrations in PBMCs were significantly increased in patients without DR but decreased in those with DR. IL-17A concentrations and Th17 cell frequency in PBMCs tended to decrease with DR severity and were negatively correlated with body mass index, T2DM duration and glycated haemoglobin. Additionally, vitreous fluid IL-17A levels were significantly elevated in patients with DR compared with those of the control group. Conclusions We conclude that disturbances in Th17 cells and IL-17A levels are possibly associated with DR.

Focal macular electroretinogram in X-linked congenital retinoschisis.

PURPOSE: To study macular function of X-linked congenital retinoschisis (CRS) by focal macular electroretinogram (MERG). METHODS: MERGs were recorded with 5 degrees, 10 degrees, and 15 degrees spots in 20 patients with CRS. Seventeen patients showed foveal schisis with little or no change in foveal fluorescein angiography (Group 1), and three patients showed advanced macular changes with nonspecific macular degeneration (Group 2). RESULTS: In Group 1, a-wave amplitudes were within the normal range, but b-waves and oscillatory potentials (OPs) had mean amplitudes significantly below those for normal control subjects. The mean b- to a-wave ratios, significantly lower than in normal eyes, decreased significantly with decreasing spot size. The implicit times of a-waves, b-waves, and OPs were significantly delayed. In Group 2, MERGs were nearly nondectable. CONCLUSIONS: The macular pathology of CSR exists mainly in the middle and inner retinal layers, disturbing the fovea more than the perifovea, whereas degeneration of photoreceptors progresses in more advanced stage.

Autosomal dominant rhegmatogenous retinal detachment associated with an Arg453Ter mutation in the COL2A1 gene.

PURPOSE: To investigate the clinical features and molecular causes of autosomal dominant rhegmatogenous retinal detachment (RRD) in two large families. METHODS: Clinical examination and linkage analysis of both families using markers flanking the COL2A1 gene associated with Stickler syndrome type 1, the loci for Wagner disease/erosive vitreoretinopathy (5q14.3), high myopia (18p11.31 and 12q21-q23), and nonsyndromic congenital retinal nonattachment (10q21). RESULTS: Fifteen individuals from family A and 12 individuals from family B showed RRD or retinal tears with minimal (family A) or no (family B) systemic characteristics of Stickler syndrome and no ocular features of Wagner disease or erosive vitreoretinopathy. The RRD cosegregated fully with a chromosomal region harboring the COL2A1 gene with maximum lod scores of 6.09 (family A) and 4.97 (family B). In family B, an Arg453Ter mutation was identified in exon 30 of the COL2A1 gene, that was previously described in a patient with classic Stickler syndrome. In family A, DNA sequence analysis revealed no mutation in the coding region and at the splice sites of the COL2A1 gene. CONCLUSIONS: In two large families with RRD, linkage was found at the COL2A1 locus. In one of these families an Arg453Ter mutation was identified, which is surprising, because all predominantly ocular Stickler syndrome cases until now have been associated with protein-truncating mutations in exon 2, an exon subject to alternative splicing. In contrast, the Arg453Ter mutation and other protein-truncating mutations in the helical domain of COL2A1 have been associated until now with classic Stickler syndrome.

Temporal changes in gene expression after injury in the rat retina.

PURPOSE: The goal of this study was to define the temporal changes in gene expression after retinal injury and to relate these changes to the inflammatory and reactive response. A specific emphasis was placed on the tetraspanin family of proteins and their relationship with markers of reactive gliosis. METHODS: Retinal tears were induced in adult rats by scraping the retina with a needle. After different survival times (4 hours, and 1, 3, 7, and 30 days), the retinas were removed, and mRNA was isolated, prepared, and hybridized to the Affymatrix RG-U34A microarray (Santa Clara, CA). Microarray results were confirmed by using RT-PCR and correlation to protein levels was determined. RESULTS: Of the 8750 genes analyzed, approximately 393 (4.5%) were differentially expressed. Clustering analysis revealed three major profiles: (1) The early response was characterized by the upregulation of transcription factors; (2) the delayed response included a high percentage of genes related to cell cycle and cell death; and (3) the late, sustained profile clustered a significant number of genes involved in retinal gliosis. The late, sustained cluster also contained the upregulated crystallin genes. The tetraspanins Cd9, Cd81, and Cd82 were also associated with the late, sustained response. CONCLUSIONS: The use of microarray technology enables definition of complex genetic changes underlying distinct phases of the cellular response to retinal injury. The early response clusters genes associate with the transcriptional regulation of the wound-healing process and cell death. Most of the genes in the late, sustained response appear to be associated with reactive gliosis.