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.
Purpose To investigate the effects of intervention with Tanakan on anterior ocular segment in diabetic retinopathy (DR) after retinal photocoagulation． Methods Prospective random controlled study was performed on 72 patients (72 eyes) with ultrasound biomicroscopy (UBM),by obtaining and quantitatively analyzing the changes of anterior ocular segment including anterior chamber, anterior chamber angle, ciliary body and choroids before and the 3rd day and the 7th day after retinal photocoagulation. Results Three days after photocoagulation, significant elev ated IOP and narrowed chamber angle were observed in control group and 4 eyes (1 1.11%) in Tanakan group (Plt;0.01)． Choroidal detachment in 32 eyes (88.89%) in control group and in 2 eyes (5.56%) in Tanakan group and the severity of ciliochoroidal detachment in tanakan group was significantly lower than that in control group． Conclusion Tanakan is effective to prevent the complications of anterior segment, such as ciliochoroidal detachment, elevation of IOP, narrowing of chamber angle occurring early after retinal photocoagulation and reduce the severity of ciliochoroidal detachment． (Chin J Ocul Fundus Dis, 2001,17:187-189)
Objective To investigate the early effects of intervention with tanakan on retinal function in diabetic retinopathy(DR) after laser photocoagulation. Methods Prospective random controlled study was performed on 60 Patients (60 eyes) from 23 to 69 years old with DR(phase Ⅲ～Ⅳ). The multifocal electroretinograms (MERG) were tested with VERIS Ⅳ before, the 3rd day and the 7th day after photocoagulation. Results No significant differences were found in the latencies and response densities of N1,P1 and N2 between the two groups before photocoagulation. Compared with that before photocoagulation, three days after photocoagulation the latencies in tanakan group had no significant change. The response densities of N1,P1 and N2 reduced and the changes were much smaller than that in control. Three days after photocoagulation, the response densities of P1 and N2 in the central macula 5°area were much higher and the latencies of P1 and N2 were significantly shorter than that in control group. There were no significant differences in the response densities in the 7th day and the differences in the latencies between two groups still existed. Conclusion Tanakan may be effective in preventing the retina from damage of retinal photocoagulation in some degree in DR. (Chin J Ocul Fundus Dis, 2002, 18: 208-211)
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells. METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml. RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml. CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration （Chin J Ocul Fundus Dis,1997,13: 86-88）
OBJECTIVE:To verify the safe dose of cephradine in intravitreal injection. METHODS:After injecting different doses of cephradine(100mu;g,200mu;g,250mu;g,300mu;g,400mu;g)into vitreous cavity of different group of rabbits the activities of the retinal enzymes (SDH,LDH )on different time (Id,3d, 7d ) were determined respectively, and the histological and ultrastructural changes of retinas were also observed simuhaneously. RESULTS:The activity of rellnal SDH and LDH was found to be decreased gradually with tbe icreasing of the dosage of intravitreal cephradine. The activities of SDH and LDH were found in the lowest level on tile 3rd and lsl day,but they recover to normal levels on tile 7th day after intravitreal in}eetion in 100mu;g,200mu;g groups,and still lower tban normal in the other groups. Histologically,retinal edema was found both in 100mu;g and 200mu;g groups,but degradation of retinal cells,and loss of cones and rods were round in the 250mu;g, 300mu;g and 400mu;g groups. CONCLUSION: The safe dose of intravitreal injection of cepbradlnc is 200mu;g. （Chin J Ocul Fundus Dis,1997,13:139-142 ）
Objective To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway. Methods Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-alpha; for 24 hours, 1 and 6 mu;g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-alpha; for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059［the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2］for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay. Results In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and b syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-alpha; or LPS increased, the fluorescence intensity decreased（Plt;0.01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected (Plt;0.001). Pretreatment with 50 mu;mol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls（Plt;0.05）. Conclusions TNF-alpha; and LPS down-regulate the expression of syndecan-1 in cultured human RPE cells. The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway. (Chin J Ocul Fundus Dis, 2006, 22: 113-116)
Objective To observe the change of diffusion upper limit of macromol ecules through pathological retina and the difference between the layers of retina. Methods Retinal edema was emulated by establishing branch retinal vein occlusion (RVO) model in miniature pig eyes under photodynamic method. Two days later, the retinas of both eyeballs were peeled off. The diffusion test apparatus was designed by ourselves. FITC-dextrans of various molecular weights (4.4, 9.3, 19.6, 38.9, 71.2 and 150 kDa) and Carboxyfluorescein (376 Da) were dissolved in RPMI1640 solutions and diffused through inner or outer surface of retina. The rate of transretinal diffusion was determined with a spectrophotometer. Theoretical maximum size of molecule (MSM) was calculated by extrapolating the trend-linear relationship with the diffusion rate. In separate experiments to determine the sites of barrier to diffusion, FITC-dextrans were applied to either the inner or outer retinal surface, processed as frozen sections, and viewed with a fluores cence microscope. Results FITC-dextrans applying to inner retinal surface, 4.4 kDa dextrans were largely blocked by inner nuclear layer (INL); 19.6,71.2 kDa dextrans were blocked by the nerve fiber layer (NFL) and inner plexiform layer; 15.0 kDa dextrans were blocked by NFL. FITC-dextrans applying to outer retinal surface, most dextrans with various molecular weights were blocked before outer nuclear layer (ONL). No matter applying to the inner or outer surface, Carboxyfluore scein can diffuse through the whole retina and aggregate at INL and ONL. After RVO, the inner part of retina became edema and cystoid, loosing the barrier function. Compared with the normal retina, the MSM in RVO tissues increased (6.5plusmn;0 39nm Vs 6.18plusmn;0.54nm, t=4.143, P=0.0001). Conclusions A fter RVO, the barrier function of inner part of retinal is destroyed and the upper limit of diffusion macromolecule size increased, which is nevertheless limited. ONL acts as bottle-neck barriers to diffusion, if the outer part of retina is damaged, the change of the diffusion upper limit will be prominent. （Chin J Ocul Fundus Dis，2008，24：197-201）
Objective To observe the protective effect on retinal ganglion cells (RGC) and the safety of intravitreal injected acteoside in rats. Methods A total of 50 male Sprague Dawley rats with the weight of 190-210 g were used in this study. Fifteen rats were used for safety experiment of intravitreal injection of acteoside. The rats were divided into group A, B, C, control group and blank group, three rats in each group. The rats in group A, B and C were received intravitreal injection of 5 mu;l acteoside at the concentration of 1, 2, and 5 mg/ml, respectively. Phosphate buffer solution (PBS) was injected in rats of control group. No treatment was performed for blank group. The retinal structure was examined by hematoxylin-eosin (HE) staining of retinal frozen sections at one, two and three weeks after injection. The retinal ultrastructure was examined by ultrathin section under transmission electron microscope at one and three weeks after injection. Others 35 rats were used for experiment of protective effect of acteoside on RGC. The rats were divided into operation group A and B (n=8), sham operation group C and D (n=8), and blank group (n=3). The optic nerve of rats in operation group was clamped for 10 seconds after optic nerve exposure, while the optic nerve of rats in sham operation group was exposed only. The rats in operation group A and B were received intravitreal injection with 5 mu;l acteoside (1 mg/ml) and 5 mu;l PBS respectively. The rats in sham operation group C and D were received intravitreal injection with 1 mu;l acteoside (1 mg/ml) and 1 mu;l PBS respectively. No treatment was performed for blank group. The retinal structure was examined by HE staining of retinal frozen sections at one, two and four weeks after injection. Immunohistochemistry was used to measure the expression of growth associated protein 43 (GAP-43). RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTPbiotin nickend labelling (TUNEL) method. Software of SPSS 13.0 was used for the data statistical analysis in this study. Results In the safety experiment of intravitreal injected acteoside, there was no abnormity of cornea, anterior chamber, lens, vitreous cavity and retina after injection. At one, two and three weeks after injection, the retina structure was normal without significant apoptosis, necrosis and inflammatory cell infiltration. The ganglion cell layer showed slightly edema; there was no obvious change of retinal ultrastructure after injection of acteoside with 5 mg/ml and 2 mg/ml, but slight change with the format of 1 mg/ml. Transmission electron microscopy showed that intravitreal injection of 5 mu;l acteoside at the concentration of 2 or 5 mg/ml can induce significant changes of micro-structures of retina, while injections at 1mg/ml can only induce minor changes.In the experiment of protective effect of acteoside on RGC, light microscope revealed that the cell showed typical changes of apoptosis in operation group, but not in sham operation group and blank group. At the first and second week after injection, compared with the sham operation group and blank group, the RGC number was decreased in operation group. The difference of RGC numbers between operation group A and B was statistically different (F=26.206,P＜0.05). The RGC numbers in operation group continues to decrease at the fourth week after injection, there was obvious difference compared with the first and second week after injection (F=17.364,P＜0.05), but there was no difference of RGC numbers among sham operation intragroup and between sham operation group and blank group at all the time points. Immunohistochemistry showed that at the first week after injection, the integrated absorbance (IA) value in operation group was higher than that in other groups (F=33.466,P＜0.05); there was no difference of IA value between operation group A and B. At the second week after injection,IA value in operation group A had slightly declined, but higher than that in operation group B (F=14.391,P＜0.05). At the fourth week after injection,IA value in operation group A declined further, but also higher than that in other groups (F=4.178,P＜0.05). TUNEL showed that on the first week after injection, RGC apoptosis rate in operation group was increased than that in other groups (F=15.365,P＜0.05). At the second week after injection, RGC apoptosis rate in operation group was decreased, and it in operation group A was lower than that in operation group B (F=15.365,P＜0.05). At the fourth week after injection, RGC apoptosis rate in operation group was decreased obviously, there was no difference compared with other groups (F=2.057,P＞0.05). There was no difference of RGC apoptosis rate between sham operation group and blank group at all the time points. Conclusion Intravitreal injection of 5 mu;l acteoside (1 mg/ml) is safe for rat retina, and can upregulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats.
Objective To investigate the retinal toxicity and verify the safe dose of intravitreal injection of nonsteroid anti-inflamatory drug,diclofenac sodium.Methods Twenty-eight healthy adult white rabbits were divided at random into 7 groups and received in every right eye the intravitreal injection of a single dose of diclofenac sodium solution ranging from 0.4-0.1 mg/0.1ml respectively ,the left eyes were regarded as conreol ones.Before injection and on the 1st,3rd,7th,14th,21st,and 28th day after injection the electroretinography on both eyes was examined.On the 28th day after injection the retinas of two rabbits of every group were examined by using light microscopy.On the 10th and 30th day after injection the retinal tissues around the optic nerve sisk of two eyes from every group at random were tested by using transmission electron microscopy.Results The retio of amplitude ofb wave of electroretinography in 0.4mg and 0.5mg groups had no sighnificant difference from groups before injection,the retinal tissues showed no structural changes in light and ecectron microscopy examination.The ratio of amplitude ofb wave of photoptic electroretinogrphy in 0.6mg groups in the early stage after injection was markedly reduced（P＜0.05）and returned to that before injection with time,reversible change of the edematou retina was discovered.The ratio of amplitude of b wave of electroretinography in 0.7-1.0mg groups was distinctly descreaded after injection（P＜0.05 or P＜0.01）,the cells of all the retinal layer revealed apparent and irreversible damage.Conclusion The largest dose of safety of intravitreal diclofenac sodium should be not more than 0.6mg.The toxic effect of intravitreal diclofenac sodium on retina is concerned mostly to cones and rods.
Objective To investigate the effect of ultra-filtration on reducing the matrix effects of the immersionof recombination human acellular dermal matrix (rhADM) on detecting residual bovine serum albumin (BSA) by ELISA.Methods Preparation of rhADM immersion: rhADM were rinsed, and then rhADM immersion were prepared. Physiologicalsal ine was used as immersion medium. Presaturation and ultra-filtration: marked the ultra-filtration tubes as PR1 (presaturation protocol 1), PR2 (presaturation protocol 2) and rhADM, respectively, added 2 mL of 1 mg/mL and 10 μg/mL BSA solution into PR1 and PR2 respectively, and added 2 mL of rhADM immersion into rhADM tubes (rhADM1 and rhADM2). The tubes were then centrifuged at 1 500 × g for 20 minutes. The above steps were repeated for 3 times. Take the inner-tube of ultrafiltration into unused centrifuge tube. Added 4 mL of 10 μg/mL BSA solution in PR1 and PR2 tubes, 4 mL of rhADM immersion in rhADM tubes, centrifuged at 1 500 × g for 20 minutes, and then the filtration was colleted. Detecting BSA concentration: the BSA concentrations of all samples were detected by using the quantitative measure of residual BSA ELISA kit. The recoveries of 10 μg/ mL BSA solution treated by presaturation protocol 1 and 2 were calculated (untreated 10 μg/mL BSA solution was as the basic sample, marked R10 and R20 respectively). The correlation coefficient between the logarithm of the filtrate dilution and the absorbance (A) value was calculated and compared with that of water exact without ultra-filtration. Results The BSA concentration of PR1 and R10 was (23.80 ± 1.58) μg/ mL and (9.04 ± 0.24) μg/mL, respectively. The BSA concentration of PR2 and R20 was (8.64 ± 0.24) μg/mL and (8.12 ± 1.01) μg/ mL, respectively. The average recovery of 10 μg/mL BSA was 263.4% ± 16.9% and 106.5% ± 3.0% when the ultra-filtration tubes were presaturaed by PR1 and PR2 (P lt; 0.01), respectively. The BSA recovery of PR2 met the detecting demand. The correlations between A value and sample dilution were increased, the correlationcoefficient was raised from — 0.727 to — 0.960 after rhADM immersion were treated by ultra-filtration. Conclusion Theresults show that the matrix effects can be reduced effectively by ultra-filtration, indicating that an acceptable recovery of BSA can be acquired when ultra-filtration tube is presaturated by sample water extract.