The Expression Of VEGF 164 And SEMA3A In The Oxygen Induced Retinopathy In Rats

ObjectiveTo observe the expression of vascular endothelial growth factor 164 (VEGF164) and Semaphorin 3A (SEMA3A) in the rat model of oxygen induced retinopathy (OIR) and to investigate their relationship in neuro-vascular changes.Methods1. The 50/10 OIR rat model:65 newborn Sprague-Dawley rats were cycled between 50% and 10% oxygen (mixed by nitrogen and oxygen) daily for 14 days and were returned to room air while 42 pups were raised in room air as controls. Flattened retinas of 16 pups from the two groups at postnatal day (P) 18 and P24 were incubated with ADP. The retinal cross-sections were stained by Hematoxylin-eosin in 10 pups of the two groups at P12, P15, P18, P21 and P24. Images of the blood vessel layers and HE stained retinal sections were captured with a microscope and digitally stored for analysis of vascular endothelia intruded into the vitreous.2. Total RNA and protein were extracted from retinas of 24 OIR pups and 24 control pups at P12, P15, P18, and P24. VEGF164 and SEMA3A mRNA and protein expression were detected by real time PCR and Western Blot respectively. Cryosection of retinas were stained by anti-rat VEGF164 or anti-rat SEMA3A antibody coupled with TRITC or FITC conjugated second antibody and then detected by confocal laser microscope.3. Randomly selected 23 OIR pups received intravitreous injections of anti-VEGF164 antibody (100ng in 1ul) in the left eyes and IgG (100ng in 1ul) in the right eyes on P12. Total RNA and protein were extracted from retinas in P18 and P24. VEGF164 and SEMA3A mRNA and protein expression were detected by real time PCR and Western Blot. Retinal localization of VEGF164 and SEMA3A were detected by immunofluorescence staining.4.3 OIR pups received intravitreous injections of VEGFab and IgG,3 OIR pups without injection as well as 3 normal pups were sacrificed at P18. TUNEL staining was used to observe the apoptosis in the retina. And the expression of Bax, Bcl-2 and Caspase-3 was detected by real time PCR. Results1. The ADPase staining showed that alternative hyper and hypo-oxygen inspiration postponed the retinal vascularization and abnormal new vessels appeared obviously at P18.2. The expression of VEGF164 and SEMA3A increased in OIR group. The transcription of SEMA3A showed some accordance with VEGF164. But the spatial changes of protein expression during growth were different from each other. VEGF164 increased and the strongest immunoreactivity was noted at P15. SEMA3A increased and the florescent density reached the peak at P24.3. The expression of SEMA3A decreased while VEGF164 was partially blocked after intravitreous injection of VEGFab. SEMA3A was obviously seen in outer plexiform lays, photoreceptor IS/OS layers.4. The expression of Bax and Caspase-3 mRNA increased in OIR retinas at P18. There was much more apoptosis in the ganglion cell layers, inner nuclear layers and outer nuclear layers in OIR rats than it in controls at P18. The ratio of apoptotic cells decreased after intravitreous injection of VEGFab as well as down regulation of Bax and Caspase-3 mRNA while up regulation of Bcl-2.Conclusions1. Alternative inspiration of 50% and 10% oxygen successfully induces the OIR modal in rats. The peak time of neovascularization is around P18.2. The expression of SEMA3A is probably influenced by VEGF164. High expression of SEMA3A in OIR rat is down-regulated after VEGF164 is partially blocked by VEGFab.3. Severe apoptosis appeares in the ganglion cell layers, inner nuclear layers and outer nuclear layers in OIR rats. VEGF164 blockage alleviates the apoptosis in OIR retinas. SEMA3A and VEGF164 probably influence the pathogenesis of neuro-vasular changes of OIR simultaneously.