| Purpose:Retinopathy of prematurity (ROP) is an ischemia-induced proliferative retinopathy,which affects premature infants with low birth weight. It is a leading cause of visualimpairment and blindness in children, and shares pathophysiological characteristics withother common ocular diseases such as diabetic retinopathy, central vein occlusion, andage-related macular degeneration. Understanding the mechanism of the ischemia-inducedproliferative retinopathy will help to develop new prevention and treatment methods forthese disorders. A mouse model of oxygen induced retinopathy (OIR) was used in thisstudy. The development of retina vasculature was observed during the first 17 days. Anunbiased quantitative proteomic approach, iTRAQ (isobaric tagging for Relative andAbsolute protein Quantification) coupled with 2D nano LC-MS/MS, was employed toestablish the mouse retina proteome and obtain differential protein expression in theoxygen induced retinopathy mouse model. The results may provide the insights into themechanism of the ischemia-induced neovascularization of retina.Methods:1. Oxygen induced retinopathy mouse model of ROP was developed in C57BL/6J miceby a5-day exposure to 80％oxygen from P7 to P12. Retina whole mounts of 8developmental stages selected from postnatal day zero (P0) to P18 mouse werestained with ADPase histo-chemistrical method or FITC dye-perfusion techniques.Retina serial sections of the P17 were used to assess the developmental growth of theretina vasculature.2. Retinas were mechanically dissected from enucleated eyes and stored in -80℃refrigerator. Protein was extracted using lysis buffer from the retina tissue. Totalprotein concentration was measured using a protein assay kit based on the BCA method (Pierce, USA).3. In the discovery stage, quantitative proteomic profile of control group (pooled retinatissue samples from 20 eyes) was compared with that of OIR group (pooled retinatissue samples from 20 eyes) to search for differential expressed proteins. The resultswere then verified using individual samples. Control group was labeled with iTRAQ114 and 115 reagents and the OIR group labeled with iTRAQ 116 and 117 reagents,respectively. The labeled samples were then pooled together and subjected tosubsequent 2D nano LC-MS/MS analysis.4. Identification of proteins was achieved using ProteinPilot software and Mascottsoftware and searched against the International Protein Index (IPI: Version 3.01)protein database. Relative quantification of proteins using iTRAQ technology isbased on the ratio of peak areas of m/z 114, 115, 116 and 117 Da from MS/MSspectra.Results:1. There are no retinal vessels in the new born mouse on the first day. Vitreous vesselsand ADPase positive large cells could be found throughout the retina. The retinalvessels take the central 1/3 of the retina on P4, and 2/3 on P10. On P12, a kind ofspiral intermediate arteries appear which connect the superficial and deep vascularplexus. There miniscent vitreous vessels do not disappear until P17, when retinalvessels final develop maturely.2. Oxygen induced retinopathy (OIR) mouse model was successfully inducedfollowing 5-day exposure to 80％oxygen. Neovascularization was observed fromP14 by ADPase staining on retina whole mounts and get to the peak on P17. It alsoconfirmed pre-retina nuclei counting in serial sections.3. In total 261 proteins were identified including 48 proteins of optical activity/signaltransduction, 4 proteins of cell cycle/death, 46 proteins of cell structure/motility/transport/traffic, 33 proteins of chromosome, 84 proteins ofmetabolism, 11 proteins of immunity/defense and 21 proteins with unknownfunction.4. The levels of 29 proteins (14 up-regulated and 15 down-regulated) showedsignificant differences between control group and OIR group. The levels of 4crystallin related proteins were found to be significantly decreased and 3 plasmaproteins increased in OIR group.Conclusions:1. OIR mouse model was a good model for the mechanism study ofvasculogenesis andangiogenesis.2. iTRAQTM technology coupled with 2D nano LC-MS/MS was successfully applied toanalyse the retina proteome.
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