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 investigate the effects of lights with different wavelength on the retina of rd12 and C57BL/6J mice. Methods Thirty two rd12 mice and C57BL/6J mice were randomly divided into the control group, white light group, midwavelength light (505 nm) group and shortwavelength light (405 nm) group, with eight mice in each group. Besides the control group, other groups were exposed to cycle illuminations ［12 hours dark, 12 hours (800plusmn;130) Lux］ for seven days to establish the model of retinal light damage. Electroretinogram (ERG) responses of all mice were recorded at the day before illumination and 1st, 4th and 7th days after illumination. The eyes were enucleated at 7th days after illumination to assess levels of reactive oxygen species (ROS), expression of peroxiredoxin 6 (PRDX6), and activity of caspase-3. Results ERG amplitudes of all groups declined gradually in C57BL/6J mice, and the most significant effects was found in the short-wavelength light group. The amplitudes of photopic b-wave were significantly different at 1st, 4th and 7th days (F=4.412, 5.082, 9.980；P＜0.01). The amplitudes of cone b-wave of the four groups decreased to (85plusmn;10) %, (70plusmn;19) %, (57plusmn;22) % and (46plusmn;19) % at 7th days, respectively, and were significantly different between white light group and short-wavelength light group(t=3.19,P＜0.01). The levels of ROS were significantly different in rd12 mice (F=16.08,P＜0.01), and elevated obviously in shortwavelength light group. The expressions of PRDX6 of retina were significantly different in rd12 mice (F=7.214,P＜0.05), and were decreased obviously in short-wavelength light group. The caspase-3 relative activity was significantly different in rd12 retina (F=7.530,P＜0.05); but there was no significant difference in C57BL/6J mice (F=3.625, 1.993, 1.133; P＞0.05).The caspase-3 relative activity were significant different between rd12 mice and C57BL/6J mice in short wavelength light group (t=5.474,P＜0.05). Conclusions Short-wavelength light can induce retinal damage of mouse retina, especially in rd12 mouse. The retinal light damage possibly relates to the oxidative damage.
Objective To invesitgate the influence of recombinant adenovirus vector of human pigment epithelium-derived factor（AV-hPEDF）on retinal new vessels mediated by recombinant adenovirus vector. Methods Twenty 7-days-old Sprague-Dawley (SD) rat were divided into two groups randomly after the establishment of retinal neovascularization model. At postnatal 14 day, they were accepted intravitreal injection with blankadenovirus-vector (AV-Blank group) and adenovirus-vector PEDF(AV-PEDF group) respectively. The retinal vascular endothelial cells were counted, the PEDF mRNA and protein expression in retina and vitreous were determined by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistochemistry. Results After injection with medicine, the number of RNV was decreased obviously in AV-PEDF group（t＝42.009，Plt;0.001）;the protein expression of retinal PEDF was increased obviously in AV-PEDF group（t=36.638，Plt;0.001）; the PEDF mRNA expression in vitreous was also increased obviously in AV-PEDF group （t=9.128，Plt;0.001）. Conclusion Recombinant Adenovirus vector mediated PEDF can raise the PEDF expression in the retinal and vitreous neovascularized tissues in rat, which suggested that the expression of PEDF may be related to inhibition and reduction of RNV.
Objective To observe the inhibitory effect of kallikrein-binding protein (KBP) on choroidal neovascularization. Methods Forty Brown Norway rats were randomly divided into the KBP groups and the control group, 20 rats in each group, the right eye as the experimental eye. The rats were photocoagulated by 532 nm laser to induce CNV model. One week after laser photocoagulation, the rats were received FFA examination. At the second day after FFA examination, the rats of KBP group were received an intravitreal injection of KBP 5 mu;l (4 mg/ml KBP). The same volume of deionized water was injected into the rats in the control group. The rats of two groups received FFA examination at one, two and three weeks after injection. The expressions of vascular endothelial growth factor and pigment epithelium derived factor were observed using hematoxylin and eosin stain and immunohistochemistry stain. CNV leakage area and the cumulative absorbance of laser spot area were analyzed by Image-Pro plus 6.0 software. Results FFA examination showed that there were CNV and fluorescence leakage at one week after laser photocoagulation; one, two and three weeks after injection, the leakage decreased gradually in KBP group, but increased with time in control group. Compared with control group, the spot area and CNV in KBP group reduced gradually, but CNV was always there in control group. The differences of VEGF (F=1.29) and PEDF (F=6.29) expressions at one week after laser photocoagulation were not statistically significant (P＞0.05). The differences of VEGF and PEDF expressions at one, two and three weeks after injection were statistically significant（VEGF:F=14.16,66.89,24.34; PEDF：F=4.22,62.04,233.05;P＜0.001）.Conclusion Intravitreal injection with KBP can inhibit CNV.
Objective：To observe the expression of gene and protein l evel of unfolded protein, glucoseregulated protein 78 (GRP78), after retinal d etachment (RD); to find out the relationship between UPR and the cell damage after RD. Methods：Eightyeight Wistar rats were divided into 2 groups: con trol group (11 rats) and RD group (77 rats). In RD group, subretinal injection with 10 mg/ml hyaluronic acid sodium was performed on the left eyes of the rats t o set up RD model, and the left eyes and retinal tissue were collected 1/2 day, 1 day, 2, 4, 8, 1 6 and 32 days after RD; there were 11 rats in each subgroup. The expression of G RP78 mRNA in retina tissue was detected by semiquantitative reverse transcript i on polymerase chain reaction (RT－PCR), the expression of GRP78 protein level wa s detected by Western blotting, and the distribution of GRP78 in each retinal lay er was observed by immunofluorescence labeling method and confocal microscopy. Results：The expression of retinal GRP78 mRNA significantly in creased in 1/2 day , 1 day, 2, and 4 days subgroups after RD (Plt;0.05). The expression of GRP7 8 protein significantly increased in each subgroup after RD compared with which in the control group, and reached the peak in 8, 16, and 32 days subgroups. The expres sion of GRP78 protein was detected in all of the retinal layers after RD. Conclusion：The protection mechanism of UPR starts up after RD, and l eads the correc t pucker of the protein and reduces cellular injury by upregulating the expres s ion of GRP78, which provide the theoretic basis for reducing the cellular injury and improving the visual function in patients with RD.
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.
Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs′ axon. 4′, 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P＜0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P＜0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P＜0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.
ObjectiveTo observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells. MethodsCultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively. ResultsThe mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05). ConclusionKnockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
Objective： To observe the effect of estrogen on the expr ession of pigment epithelium derived factor (PEDF) in cultured retinal Muuml;ller cells under the anoxic condition． Methods：After the anoxic retinal Muuml;ll er cells were tre ated with estrogen (E2) with the concentration of 10-6、10-5 and 10-7 mmol/L, t he level of expression of PEDF mRNA and the protein was detected by reverse tran scriptionpolymerase chain reaction and Western blotting analysis. Results：Th e expression of PEDF mRNA and protein decreased 24 hours after anoxia. E2 with t he concentration of 10-5 and 10-6 mmol/L inhibited the decrease of expression of PEDF mRNA and protein induced by anoxia, which related to the concentration of E2． Conclusion：strogen can regulate the expression of PEDF, which ma y play an important role in the regulation of retinal neovascularization．