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 effect of visible light on apoptosis of cultured human retinal pigment epithelium (RPE) cells. Methods Being the light source,500lx,(2 000±500)lx and (3 400±200)lx cold white light were used. The duration of exposure was 0,6,12 and 24 hours respectively. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Annexin V-flunorescein isothiocyanate/Propidium iodium labelling and flow cytometry. Results Apoptosis and necrosis were found in cultured human RPE cells which were exposed to visible light.(1)A significant increase in apoptotic and necrotic percentages was consistent with a higher light intensity.(2)Apoptosis was the main response to shorter (6 h and 12 h) exposure duration,while necrosis was more pronounced correlated to the prolongation of post-exposure culture (P＜0.05),and the longer the post-exposure period was, the more apoptotic necrosis were seen.Thirty-six hours after exposure the necrotic percentages were more pronounced (P＜0.01). Conclusions Visible light (＞500 lx) increases the proportion of apoptosis and necrosis of human RPE cells in vitro.The extent is related to exposure intensity and duration. It demonstrates that the lower intensity and the shorter duration of exposure to light are, the more pronounced apoptotic percentages are observed,otherwise necrosis. (Chin J Ocul Fundus Dis, 2002, 18: 227-230)
Objective To study the response of the retinal neuronal adaptive system to changes of background illumination (BG) by measuring the oscillatory potentials (OPs) and the a- and b-waves of the electroretinogram (ERG) in different BG illuminations. Methods The a- and b-wave and the digitally filtered OPs were simultaneously recorded from Wistar Fu rats aged from 25 to 29 days during dark adaptation (DA) and during 6~8 minutes of BG illuminations at four levels increased successively by steps of two log units, i.e., ldquo;low scotopicrdquo; level of 1.43times;10-6cd/m2, ldquo;high scotopicrdquo; of 1.43times;10-4cd/m2 , ldquo;low mesopicrdquo; of 1.43times;10-2cd/m2 and ldquo;high mesopicrdquo; of 1.43times;10-2cd/m2. Full field stimulus flashes of 75 msec duration and 1.43times;10-2cd/m2intensity was delivered at an interval of 1 minute. Results Five OP wavelets were recorded in DA and during scotopic BG illuminations. The number of wavelets was reduced to three as the eyes were exposed to mesopic BG levels. However, the sum of OPs amplitudes (SOPs) increased as the BG was intensified, except at ldquo;high mesopicrdquo; level, by which a significant decrease of SOPs occurred. The amplitudes of the a-and b-waves remained unchanged at the two scotopic BG and decreased as the BG intensity increased to mesopic levels. Conclusion The response of retinal neural adaptive system of the Albino rat to changes of BG light is more sensitive and robust than the slow components of the ERG. The enhancement of the oscillatory responses at ldquo;low mesopicrdquo; illumination level suggests that using proper BG light may be conducive to reducing the variation of OPs. (Chin J Ocul Fundus Dis, 2001,17:286-288)
ObjectiveTo investigate the effect of blue light on Ca2+-protein kinase C (PKC) signaling pathway in human retinal pigment epithelial (RPE) cells in vitro. MethodsPrimary human RPE cells were cultured in vitro and characterized. The experiments were carried out using the 4th generation of human RPE cells. The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression. Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells. Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W, 450-500 nm wavelength, (2000±500) Lux], and 24 hours prolongation of post-exposure culture. The human RPE cells were randomly divided into 5 groups. Group A did not receive light irradiation, group B only received blue light irradiation, group C was blue light irradiation and 0.1 mmol/L nifedipine treatment, group D was blue light irradiation and 100.0 nmol/L calphostin C treatment, group E was blue light irradiation and 100.0 nmol/L PMA treatment. Intracellular Ca2+ concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging. ResultsThe PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1, 1.0, 10.0, and 50.0 nmol/L PMA-treated groups, the difference was statistically significant (F=217.537, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P=0.072). The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0, 25.0, 50.0, and 75.0 nmol/L calphostin C-treated groups, the difference was statistically significant (F=164.543, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C-treated groups (P=0.385). PKC level in blue light group was higher than non-light group, the difference was statistically significant (t=-9.869, P<0.05). The Ca2+ fluorescence intensity values in group B, C, D and E was higher than group A, the difference was statistically significant (F=26 764.92,P<0.05). The Ca2+ fluorescence intensity values in group E was higher than group B, C and D (P<0.05), and that in group B was higher than group C and D (P<0.05). ConclusionsThe PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue-light irradiation. Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells. PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation.
Objective To explore the protective mechanism and effect of coenzyme Q10 on the retinal photic injury in experimental rats. Methods Thirty Sprague-Dawley rats were divided randomly into 3 groups: normal control group, positive control group, and coenzyme Q10 group. The experimental model of photic injury in rats was established by being exposed to intense green fluorescent light with an illuminance level of (2000plusmn;120) Lux for 24 hours. The physiological saline and coenzyme Q10 were given through tail intravenous injection at 24 hours and 30 min before light exposure in positive control group and coenzyme Q10 group, respectively. Ophthalmectomy was performed 1 day after the illumination. Changes of retinal histopathology and ultrastructure were observed by light and electron microscope. The apoptosis rate of retinal cells was detected by flow cytometry. Results The result of histopathological examination showed that in coenzyme Q10 group, the outer segments arranged trimly with only few cell apoptosis; the inner and outer segments slightly swelled, and compared with the positive group, the histopathological changes alleviated obviously. The result of flow cytometry revealed that the apoptosis rate of retinal cells was (1.65plusmn;1.48)% in normal control group, (25.83plusmn;2.92)% in positive control group, and (12.43plusmn;2.25)% in coenzyme Q10 group, respectively. The apoptosis rate of retinal cells was higher in positive control group than that in the normal control group (t=18.28, Plt;0.01), and lower in coenzyme Q10 group than that in the positive control group (t=9.07, Plt;0.01). Conclusion Coenzyme Q10 plays an important role in preventing the photic injury of retina and optic cell apoptosis. (Chin J Ocul Fundus Dis, 2007, 23: 122-125)
Purpose To observe the pathologic changes of retinal photic injury in mice. Methods A light damaged trunk was designed by ourselves.The mice were given an intermitent light exposure for 3 days,12 hours light exposure every day and 12 hours dark adaption before every exposure.Experimental animals were sacrificed on the 1st,6th,12th,18th and 30th day after light injury,and the eyes were enucleated for light and electronic microscopy observation. Results The early pathologic changes including disc membrance swelling,disorganization in outer segments,and mitochondrial swelling,spherical change in inner segments．Then the chromatin densification,liquefaction and margination,and the shrinkage of nuclear membrance were found in the nuclear layer.Finally the outer nuclear layer became thin and disappeared.The apical microvill of RPE cell disappeared and basic fold became flat in some samples. Conclusion The photoreceptor degeneration was the pathologic characteristic of retinal photic injury in mice. (Chin J Ocul Fundus Dis,1998,14:215-218)
Objective To assess the protective effect of recombinant human erythropoietin (EPO) on human retinal pigment epithelial (RPE) cells injured by light. Methods Cultured human RPE cells were exposed to light for 12 hours, and the culture was stopped 24 hours later. The 3(4,5dimethylthiazole2y1)2,5diphenyl tetrazolium bromide (MTT) cell viability assay and annexin V flunorescein isothiocyanate/propidium iodium labeling and flow cytometry were used to assess the effects of EPO with different concentration on the cellular viability and apoptosis of human RPE cells. The protective effect and mechanism of EPO on RPE cells injured by light was detected by adding AG490. Results EPO, especially with the concentration of 40 IU/ml, obviously increased the cellular viability of RPE cells and apparently decrease the cellular apoptosis induced by light injury. After adding AG490, the effects of EPO on cellular viability and apoptosis were inhibited. Conclusion It is suggested that EPO can protect the human RPE cells from lightinduced injures, and its protective mechanism works after the combination of EPO and its receptor.
Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.
Objective To assess the effects of 670nm LED (lightemitting diode) to protect the photoreceptor from the lightinduced damage in a rat model. Methods 32 SD rats were randomly assigned to one of eight groups: untreated control group, the LEDtreated control group, three groups of lightinduced damage，and three groups of lightinduced damage treated with LED. Lightinduced damage result from exposing to constant light for 3 hours of different illuminations of 900，1800 and 2700 lx, respectively. The LED treatment (50 mW) was delivered for 30 minutes at 3 hours before the light damage and 0，24 and 48 hours after the light damage. Retinal function and morphology were measured by electroretinogram (ERG) and histopathology assay. Results The illumination of 900 lx for 3 hours did not damage the rat retina. The illumination of 1800 lx for 3 hours resulted in thinner ONL and no OS and IS. The ratio of damaged area/total retinal area was 048plusmn;012， the damaged thickness of ONL/normal ONL (L5 ) was 039plusmn;007，and the amplitude of ERG b wave was (431plusmn;120) mu;V. With the LED treatment the ratio of damaged area decreased (M6=017plusmn;0.12, P5/6=0.002), and the ratio of the damaged thickness of ONL also decreased (L6=0.22plusmn;0.09, P5/6lt;0.01), and the amplitude of ERG b wave increased to (1011plusmn;83) mu;V（P5/6lt;0.001）. The illumination of 2700 lx for 3 hours caused severed damage to the rat retina and the LED could not protect them significantly. Conclusions 670 nm LED treatment has an evident protective effect on retinal cells against light-induced damage, which may be a simple and effective therapy to prevent or to delay agerelated macular degeneration.
Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.