ObjectiveTo observe the inhibitory effect of intraocular injection of recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor (rAAV-PSF) in oxygen induced retinopathy mice model. MethodsEighteen C57BL/6J mice were divided into 3 groups randomly, including normal group, rAAV-PSF injection group, rAAV injection group. Western blot analysis was applied to detect the transfection expression level of PSF. The other 48 C57BL/6J mice were randomly divided into 4 groups: normal group, ischemia-induced retinopathy (OIR) group, rAAV-PSF treated OIR group, rAAV treated OIR group, 12 mice in each group. Placed all mice (excepted the mice in control group) in cages of (75±2)% oxygen concentration environment for 5 days then moved to a normal environment for 5 days to induced the OIR model. At the 12th day the OIR rAAV-PSF treated OIR group was intravitreal injected with 2 μl 5×1013 pfu/ml rAAV-PSF and the rAAV treated OIR group was treated with intravitreal injection of 2 μl 5×1013 pfu/ml rAAV. The mice in OIR group were left intact after moved out of oxygen cages. Five days after injection, the eyeballs were harvested and retinal sections were stained to count the nuclear of retinal endothelium cells. Western blot analysis was applied to detect the protein level of vascular endothelial growth factor (VEGF) in retina. ResultsThere was a significant difference of the expression PSF between normal group and rAAV-PSF treated group (F=16.05,P=0.001). There was no significant difference of the PSF expression between normal group and rAAV treated group(F=16.05,P=0.890). There was a significant difference of the number of retinal endothelium cells nuclear between normal group and OIR group (F=101.00,P=0.007). There is a significant difference of the number of retinal endothelium cells nuclear between rAAV-PSF treated group and OIR group (F=101.00,P=0v002). There was no significant difference of the number of retinal endothelium cells nuclear between OIR rAAV treated group and OIR group (F=101.00,P=0.550). There was a significant difference of VEGF protein expression between normal group and OIR group (F=13.20,P=0.005), OIR group and rAAV treated OIR group (F=13.20,P=0.001). There was no difference of VEGF protein expression between OIR rAAV treated group and OIR group (F=13.20,P=0.071). ConclusionThe rAAV-PSF vitreous injection can inhibit the expression of VEGF in OIR mice, hence to restrain the proliferation of neovascularization.
ObjectiveTo study morphological characteristics and microRNA (miR) expression profiling in a mouse model of oxygen-induced retinopathy (OIR). MethodsHealthy C57BL/6J female mice and pups were randomly divided into normal and OIR group at postnatal day 7 (P7). The normal group was raised in a conventional cage and exposed to room air for 10 days. The OIR group was raised in a sealed chamber and exposed to (75±2)% oxygen. The moms were alternated between the two groups every day to promote their survival under hyperoxia. The OIR group was returned to the room air at P12. At P17, mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran), the eye balls were fixed in 4% paraformaldehyde, and the retinal whole mounts were prepared. The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope; the eye balls were also processed for paraffin sections and Hematoxylin and Eosin (H&E) staining. The cell nucleus in the newly-formed vessels beyond the inner limiting membrane was quantified. The miR was extracted from the eyes, reverse transcribed, and subjected to a customized miR array analysis. The real-time PCR was preformed to verify the results of the miR array. ResultsRetinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the avascular area was prominent in central retina. In contrast, the vessels were smooth, organized, and evenly distributed in the retinas of normal group. The percentage of avascular area in total retina area in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P < 0.01, normal group vs OIR group). H & E staining showed that the number of the cell nuclei beyond inner limiting membrane was (28.41±4.01) in OIR retina, which was substantially higher than that (0.16±0.31) in normal retina (P < 0.01, normal group vs OIR group). More interestingly, the results of miR array showed that 21 out of the 80 miRs examined exhibited more than 1.5-fold changes at expression level. Among these 21 miRs, 9 were up-regulated, 12 were down-regulated; 4 miRs showed more than 3-fold expression changes, 3 were down-regulated and 1 was up-regulated. The expression of the 4 miRs was verified by real-time PCR. The expression trends of miR-3078, miR-140, miR-29b and miR-29c were consistent with those revealed by the miR array. MiR-3078 was significantly up-regulated (t=-2.380, P < 0.05. normal group vs OIR group), and the other 3 miRs were significantly down-regulated (t=2.638, 2.323, 2.415, P < 0.05. normal group vs OIR group). ConclusionsThe OIR mouse model has been established in our study. Differential expression of the microRNAs, including miR-3078, 140, 29b and 29c, was detected in normal and OIR mouse retinas. These miR expression changes may be associated with retinal neovascularization. These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy.
ObjectiveTo investigate the efficacy of laser photocoagulation and intravitreal ranibizumab treatment of retinopathy of premature(ROP). MethodsThis study included 49 ROP infants (96 eyes), including type 1 pre-threshold ROP (7 infants, 14 eyes), threshold ROP (38 infants, 44 eyes) and aggressive posterior ROP (AP-ROP, 4 infants, 8 eyes). According to the treatments received, all patients were divided into laser photocoagulation (LP) group (40 infants, 78 eyes) and intravitreal ranibizumab (IVR) treatment group (9 infants, 18 eyes). Generally, zoneⅡand stage 3 ROP with clear refractive media received laser photocoagulation, zoneⅠROP and AP-ROP, or eyes with unclear refractive media or infants with poor general condition received IVR. The infant gestational age, birth weight, corrected gestational age at first treatment and the cure rate of the first treatment were analyzed between the two groups, and between three disease types (type 1 pre-threshold, threshold and AP-ROP). ResultsThe gestational age and birth weight was no difference between the LP group and IVR group (t=0.827, 1.911; P > 0.05). The corrected gestational age at first treatment of LP group was significantly smaller than that in the IVR group (t=3.041, P < 0.05). In the LP group, 75 of 78 eyes (96.15%) was cured by the first treatment, 3 of 78 eyes (3.85%) progressed to stage 4A after the first treatment and was controlled by vitrectomy. In the IVR group, 8 of 18 eyes (44.44%) was cured by the first treatment, 10 of 18 eyes (55.56%) progressed to next stage after the first treatment and was controlled by additional laser photocoagulation or repeated IVR. The gestational age and birth weight was no difference between type 1 pre-threshold, threshold and AP-ROP infants (t=2.071, 0.664; P > 0.05). The corrected gestational age at first treatment of type 1 pre-threshold infants was the same of the threshold lesion infants (t=2.054, P > 0.05). The corrected gestational age at first treatment of AP-ROP infants was significantly smaller than that of type 1 pre-threshold and threshold lesion infants (t=3.250, P < 0.05). The cure rate was statistically significant (χ2=24.787, P < 0.05) between there three ROP lesions. ConclusionIVR treatment is suitable for zoneⅠlesions, AP-ROP and Plus lesions, while laser photocoagulation is appropriate for zoneⅡlesions with fibrosis and less vascular proliferation.