ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage.
Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted.
Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05).
Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
Objective To observe the effects of immunologic cytokines or anti-angiogenesis gene transfer mediated by electroporation for choroidal melanoma cells.Methods The human embryo kidney cells and malignant choroidal melanoma cells were transfected with plasmids pNGVL-mIL2, pNGVL-mIL12, pCI-sFLK-1, pCR3.1-antiVEGF121,pCI-ExTek. Then the expression of mIL2, mIL12, sFLK-1, VEGF and ExTek were detected by enzymelinked immunosorbentassay (ELISA) and Western blot. Nude mice models of malignant choroidal melanoma were established and they were divided into four groups randomly. Each group was treated with 30 mu;l of 0.9% NaCl, 30 mu;g pNGVL, 30 mu;g antiVEGF121+sFLK-1+ExTek and 30 mu;g mIL2+mIL12 respectively by electroporation. Seven，14, 21, 28, 35 and 42 days after treatment, the tumor volumes were measured to calculate the tumor inhibition rate. Results ELISA and Western blot showed that mIL2，mIL12，sFLK-1 and ExTek were expressed after electroporation，VEGF expression was decreased remarkably. After treatment，the tumors of mIL2+mIL12 group were greatly inhibited with a tumor inhibition rate of 97.33%，while the tumors of antiVEGF121+sFLK-1+ExTek and pNGVL group were partially inhibited with tumor inhibition rates of 53.33% and 36.33% respectively.Conclusions Immunologic cytokines transfer mediated by electroporation can inhibit the growth of melanoma，but anti-angiogenesis only have a mild effects.
Objective To establish a rat model of blood ocular barrier breakdown induced by anterior segment intraocular analogic surgery. MethodsOne hundred and fifty healthy adult male rats were randomly divided into control group and model group, 75 rats in each group. The rats were anesthetized with 1 ml/kg ketamine hydrochloride/xylazine hydrochloride solution. Three way pipes were attached to a phosphate buffer infusion bag and two intravenous catheters. One catheter was inserted 30° obliquely through the transparent cornea anterior to the limbus into the rat's anterior chamber. Then the needle was withdrawn and the sheath was indwelling. Another catheter was connected with a manometer. Intraocular pressure was varied from 0 to 12 mm Hg (1 mm Hg=0.133 kPa) 60 times, 30 times per min. The catheter was removed. The eyes were treated with ofloxacin ophthalmic solution after surgery. The 1st, 2nd, 3rd, 5th and 7th day after surgery, the integrity of the blood ocular barrier was assessed by immunohistochemical staining for albumin and quantitative measurement using Evan′s blue as a tracer. ResultsAlbumin immunohistochemical staining of the control group was confined to the iris and retinal blood vessels. The choroid was stained at each time point after surgery. Albumin immunohistochemical staining of the model group was abundant around the iris and the retinal vasculature on the 1st day after surgery. The albumin diffused throughout the iris and the retina on the 2nd and the 3rd day after surgery. The albumin reached the retinal vessels on the 5th and 7th day after surgery. The aqueous humor Evans blue leakages of the model group were higher than those of the control group on the 1st, 2nd, 3rd and 5th day after surgery. The differences were statistically significant (t=25.781, 37.433, 25.150, 19.171； P<0.01). The Evans blue leakage of the model group was close to that of the control group on the 7th day after surgery. The difference was no statistical significant（t=1.303, P=0.209）. The retinal Evans blue leakages of the model group were higher than those of the control group on the 1st, the 2nd and the 3rd day after surgery. The differences were statistically significant (t=11.997, 14.622, 23.014; P<0.01). The Evans blue leakage of the model group was close to those of the control group on the 5th and 7th day after surgery. The differences were not statistically significant（t=2.027, 0.756; P=0.058, 0.459）. Conclusion This study establishes a rat model of blood ocular barrier breakdown induced by imitating the injury to the anterior segment during intraocular surgery.
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.
Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene.Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Nontransfected NPE cells were set as control group.The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively.Results The opticin mRNA expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were decreased compared with the control group (F=10.239，P＝0.000)；the inhibitory rate were 85.7%，62.87%，54.87% and 48.77% respectively.The opticin protein expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were also decreased compared with the control group (F=17.870，P=0.000)；the inhibitory rate were 78.7%，34.6%，31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
ObjectiveTo investigate the effect of heat shock protein B8 (HspB8) downregulation on retinal ganglion cell (RGC) and retinal function in the mice model of optic nerve injury (ONC).MethodsAdeno-Associated Virus (AAV) 2 AAV2-shHspB8-GFP was constructed to knockdown HspB8. 66 adult male C57/BL6 mice were randomly divided into the control group, the ONC group, the AAV2-shHspB8 group, the ONC+AAV2-shHspB8 group, and the ONC+AAV2- GFP group. There were 10, 20, 16, 10 and 10 mice respectively, and both eyes were used as experimental eyes. Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC. By GFP immunofluorescence staining, the efficacy of AAV2-shHspB8-GFP transfer was accessed. Moreover, it was possible to identify functional and RGC survival differences between groups by optomotor response (OMR), dark adapted full-field flash electroretinogram (ff-ERG), oscillatory potentials (OPs), photopic negative response (PhNR) and retinal flat-mount RGC counting 5 days after ONC. Comparisons between two groups were made using Mann-Whitney U test, unpaired t-test, unpaired t-test with Welch’s correction, one-way ANOVA, and Bonferroni t test.ResultsCompared with the control group, the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased, and the difference was statistically significant (F=43.63, P＜0.01). Compared with the control group, the ONC group showed obviously lower visual acuity (P＜0.01), lower a-wave, b-wave, OPs, PhNR amplitude, longer b-wave latency (P＜0.05), and the survival rates of RGC in ONC3 group, ONC5 group and ONC7 group decreased in a time-dependent manner(F=384.90, P＜0.01). Transfection of AAV2 efficiency was highest on 4 weeks after IVT. Besides, there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity, ff-ERG, OPs, PhNR and RGC survival (P>0.05). In comparison of the control group, we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated (F=10.62, P＜0.01).ConclusionsExpression of HspB8 on the retina can be induced by ONC. The investigation of RGC counting, visual acuity, and ff-ERG revealed that optic nerve injury destructed functionality of mice retina and resulted to RGC death ultimately. The Most crucial finding of this research is that HspB8 knockdown had a neuroprotective effect in RGC after ONC.
Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFlt-1 on retinal neovascularization (RNV).Methods Recombinant lentivirus sFlt-1（2-3）and sFlt-1（2-4）expressing the sFlt-1 (2-3) and (2-4) immunoglobulinlike regions of sFlt-1 were constructed. 96 seven-day-old C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1: normal control; group 2: experimental control; group 3: sFlt-1（2-3）; group 4: sFlt-1（2-4）.The mice in group 2-4 were exposed to hyperoxia with （75plusmn;2）% O2 for 5 days and then returned to normoxia with 21% O2;the mice received an intravitreal injection with 1 mu;l virus of empty vector, sFlt-1（2-3）,or sFlt-1（2-4）,respectively. Five days later, all mice underwent perfusion fluorecein angiography and retinal wholemont was made to observe the changes of retinal vessels; retinal sections were stained by hematoxylin and eosin and RNV endothelium cell nucleus were counted; vascular endothelial growth factor(VEGF) and VEGF receptor-2 (KDR/Flk-1) protein were measured by Western blot.Results Seventeen days after birth, the retinal area of fluorescein leakage and RNV, RNV nucleus which breaking through inner limiting membrane in group 3 and 4 were smaller or less than that in group 2（P＜0.01）; while VEGF protein didnprime;t changed much （P＞0.05）the expression of KDR/Flk-1 decreased significantly (P＜0.01). Conclusion sFlt-1（2-3）and sFlt-1（2-4）can inhibit the formation of oxygen-induced RNV,the former virus has a better effect.
