Abstract:
Background: Proliferation of tissues is a normal feature of a developing eye. However, in adults certain pathological conditions are associated with de novo formation of tissue and thickening of certain retinal layers. The inner limiting membrane (ILM), the layer found between the vitreous body and the retina, is prone to changes in a number of ocular conditions. This membrane changes in composition with either age or pathological conditions resulting in the ILM becoming thicker, which can result in a decrease in visual acuity (VA). In certain pathological conditions there may also be epiretinal membranes (ERM) affecting VA. Experimental data has shown that these static barriers, thickened ILM and ERM, causes drug intervention to the posterior of the eye to be ineffective which makes identification of the composition of the ERM and ILM essential to allow for development of a better drug delivery system. Objective: To identify techniques suitable for the analysis of small biopsy samples and systematically characterise all the non-enzymatic protein modifications in the ILM, specifically phosphorylated proteins, small and high molecular weight proteins. To approach understanding of the composition and development of these retinal layers three techniques were adapted and tested on ILM and ERM. Method: Matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) was employed to identify small molecules with the matrix of 9-aminoacridine (9AA) and peptides, proteins and lipids with α-cyano-hydroxycinnamic acid (CHCA). Samples were also collected and reacted with antibodies in a microarray assay to identify protein composition. A whole mount procedure was done using glial fibrillary acidic protein (GFAP), 4', 6'-diamino-2-phenylindole (DAPI), glutamine synthetase (GS), phalloidin and THY-1 to label the sites of glia, nuclei, Müller's endfeet, actin and ganglion cells respectively. TUNEL assay was done to assess for cell death. Results: MALDI-TOF identified proteins of low mass to charge ratio (m/z) in ILM and ERM samples. In the whole mount immunocytochemistry, DAPI labelling in the ILM colocalised with GFAP and GS in the ILM samples. Other ILM samples labelled with DAPI showed some co-localisation with the ganglion cell marker. There was no reaction with the actin marker phalloidin. TUNEL assay showed that there were dying cells in the ILM. The antibody microarray showed expression of more than 100 proteins involved in diverse molecular pathways but in the ILM tissue most abundant were proteins of the apoptotic and cell proliferation pathway. In contrast, highly expressed proteins in the ERM were anti apoptotic molecules. Conclusions: Whole mount immunohistochemistry showed that the ILM has an expected cellular composition of microglia, ganglion cells and dying cells in addition to cells of unknown origin, likely to be inflammatory cells detected by markers in the microarray analysis. The microarray method made it possible to characterise small ILM and ERM samples easily. Expression of molecules of cell death and division pathways were found in these samples as well as evidence of hormones, and this suggests that a number of these proteins could be specific targets for an effective intravitreal therapeutic approach.