| OBJECTIVE:To establish the mice model of oxygen induced retinopathy (OIR), and evaluated the model overall; To explore the expression regulation and significance of transforming growth factor betal (TGF-β1) and Smad4 in the retina of this model for examining pathogenesis and therapeutic intervention for the retinal neovascularization.METHODS:7-day-old C57BL/6J mice of clean grade were randomized into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, mice were exposed to 75% oxygen for 5 days and then to room air; in control group, mice were raised in room air. Eye samples of mice from each group were excised at postnatal days 7 (Postnatal days 7, to express P3, the same below),12,14,17,22,25, and 30 respectively. Eye samples of P12, P14 and P17 were used to assess the developmental growth of the retina vasculature, the ones of P17, P22, P25 and P30 were used to observe the interstitial fibrosis of the retina, and the ones of P7, P12, P14 and P17 were used to examine the expression of TGF-β1/Smad4 in mice retina. The morphologic changes of retinal vessels were estimated by observing the vascular pattern in ADPase stained retina flatmounts, and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane by HE stain, using VG stain and immunohistochemical stain to assess the interstitial fibrosis of the retina. The expression of TGF-β1 in retina tissues were analyzed by immunohistochemical techniques, and also examined the expression of TGF-β1/Smad4 messenger ribonucleic acid (mRNA) in mice retina by real-time fluorescence quantitative reverse transcription polymerase chain reaction (QRT-PCR).RESULTS:1. An oxygen induced retinopathy (OIR) in mice was successfully induced following 5-day exposure to 75% oxygen, and the quantitative results of the retinal neovascularization showed: there was a mean of 33 neovascular nuclei per cross-section extending into the vitreous in hyperoxia compared to less than 1 nuclei in the normoxia control (P< 0.0001).2. Exploring the situation of the retina fibrosis:The retinal neovascularization began to fade after P17; the results of VG stain and immunohistochemistry stain were all negative.3. The expression of TGF-β1 protein and (TGF-β1/Smad4)mRNA:Immunohistochemistry stain showed that TGF-β1 staining mainly located in the internal limiting membrane at P7-P17 in control group, and the expression was decreased with individual development gradually. The expression of TGF-β1 was higher significantly in hyperoxia mice than those of control mice at the same time (P< 0.0001), with P7 exception. QRT-PCR showed that no significant difference between the two groups at P7. The expression of TGF-β1mRNA reached the peak value at P12, decreased slightly at P14, and also kept the higher level till P17 in the oxygen-induced retinopathy group. It had the highest expression at P7 in the control group, and decreased at P7-P17. With the high expression of TGF-(31mRNA after hyperoxia-induced retina injury, the expression of Smad4mRNA also increased at P7-P17. The expression of Smad4mRNA decreased at P7-P17 in the control group.CONCLUSIONS:The mice model of OIR duplicated preferably the acute pathological changes of retinopathy of prematurity (ROP) such as angiemphraxis and retinal neovasculatization, but it did not present the serious pathological changes such as retinal fibrosis and retinal detachment of ROP. It may be one of the signal transduction pathways that TGFβ1 took part in the neovascularization in retina through Smad4. |
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