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

<div><p>Purpose</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>The expression levels of interleukin 6 (<i>IL6</i>), tumor growth factor B2 (<i>TGFB2)</i>, vascular endothelial growth factor A (<i>VEGFA</i>), chemokine C-X-C motif ligand 1 (<i>CXCL1</i>), v-rel avian reticuloendotheliosis viral oncogene homolog A (<i>RELA</i>), glial fibrillary acidic protein (<i>GFAP</i>), and tenascin C (<i>TNC</i>) 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.</p><p>Conclusions</p><p>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 (<i>IL6</i>, <i>TGFB2</i>, <i>VEGFA</i>, <i>CXCL1</i>, <i>RELA</i>, <i>GFAP</i>, and <i>TNC</i>).</p></div

Expression Levels of Cytokines and Other Investigated Genes in the Solid Component of the Irrigation Solution.

<p>Expression Levels of Cytokines and Other Investigated Genes in the Solid Component of the Irrigation Solution.</p

Demographics of Patients Included in Macular Hole and Idiopathic Epiretinal Membrane Groups.

<p>Demographics of Patients Included in Macular Hole and Idiopathic Epiretinal Membrane Groups.</p

Expression Levels of Cytokines and Other Investigated Genes in the Solid Component of the Irrigation Solution.

<p>Expression Levels of Cytokines and Other Investigated Genes in the Solid Component of the Irrigation Solution.</p

Spearman’s Correlation Coefficient Analysis of the Expression Levels of Seven Genes in Eyes with Idiopathic Epiretinal Membrane.

<p>Spearman’s Correlation Coefficient Analysis of the Expression Levels of Seven Genes in Eyes with Idiopathic Epiretinal Membrane.</p

Spearman’s Correlation Coefficients between the Seven Upregulated Genes and Preoperative Visual Acuity of Eyes with Idiopathic Epiretinal Membrane.

<p>Spearman’s Correlation Coefficients between the Seven Upregulated Genes and Preoperative Visual Acuity of Eyes with Idiopathic Epiretinal Membrane.</p

Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

<div><p>Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.</p> </div